JP6507522B2 - Improved Fc binding protein, method for producing the protein and antibody adsorbent using the protein - Google Patents

Description

  The present invention relates to Fc binding proteins with affinity to immunoglobulins. More specifically, the present invention relates to an Fc binding protein having higher stability to heat and acid than wild-type, a method for producing the protein, and an antibody adsorbent obtained by immobilizing the protein on an insoluble carrier.

  Fc receptors are a group of molecules that bind to the Fc region of immunoglobulin molecules. Each molecule recognizes a single or the same group of immunoglobulin isotypes by a recognition domain on an Fc receptor by a recognition domain belonging to the immunoglobulin superfamily. This determines which accessory cells are driven in the immune response. Fc receptors can be further divided into several subtypes, such as Fcγ receptors that are receptors for IgG (immunoglobulin G), Fcε receptors that bind to the Fc region of IgE, Fcα receptors that bind to the Fc region of IgA, etc. There is. In addition, each receptor is further finely divided, and the presence of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb has been reported as Fcγ receptors (Non-patent Document 1).

Among Fcγ receptors, FcγRIIIa is present on cell surfaces such as natural killer cells (NK cells) and macrophages, and is an important receptor involved in ADCC (antibody-dependent cellular cytotoxicity) activity which is important among human immune mechanisms. It is. It is reported that the affinity between this FcγRIIIa and human IgG is 10 ⁷ M ⁻¹ or less as a binding constant (KA) indicating the strength of binding (Non-patent Document 2). The amino acid sequence of human FcγRIIIa (SEQ ID NO: 1) is published in public databases such as UniProt (Accession number: P08637). In addition, the structural functional domain of human FcγRIIIa, the signal peptide sequence for penetrating the cell membrane, and the position of the cell transmembrane region have been similarly published. FIG. 1 shows a schematic structural view of human FcγRIIIa. The amino acid numbers in FIG. 1 correspond to the amino acid numbers described in SEQ ID NO: 1. That is, from the 1st methionine (Met) to the 16th alanine (Ala) in SEQ ID NO: 1 is the signal sequence (S), and from the 17th glycine (Gly) to the 208th glutamine (Gln) is the extracellular region (EC), valine at position 209 (Val) to valine at position 229 (Val) has a transmembrane domain (TM) and lysine at position 230 (Lys) to lysine at position 254 (Lys) has an intracellular region (C) ). It is known that FcγRIIIa strongly binds particularly to IgG1 and IgG3 among human IgG subclasses ranging from IgG1 to IgG4, but weakly binds to IgG2 and IgG4.

Takai. T. , Jpn. J. Clin. Immunol. , 28, 318-326, 2005. J. Galon et al., Eur. J. Immunol. , 27, 1928-1932, 1997

  An object of the present invention is to provide an Fc-binding protein which is particularly improved in stability to heat and acid, a method for producing the protein, and an antibody adsorbent using the protein.

  As a result of intensive studies to solve the above problems, the present inventors have identified an amino acid residue involved in the improvement of stability in human FcγRIIIa, and a variant in which the amino acid residue is substituted with another amino acid residue is They have found that they have excellent stability to heat and acid, and have completed the present invention.

That is, the present application includes the embodiments described in the following (A) to (Q):
(A) Among the amino acid sequences described in SEQ ID NO: 1, which comprises the 17th to 192nd amino acid residues, and in the 17th to 192nd amino acid residues, among the following (1) to (40): An Fc binding protein, wherein at least any one amino acid substitution has occurred.
(1) The 18th methionine of SEQ ID NO: 1 is substituted with arginine (2) The 27th valine of SEQ ID NO: 1 is substituted with glutamic acid (3) The 29th phenylalanine of SEQ ID NO: 1 is substituted with leucine or serine (4) The 30th leucine of SEQ ID NO: 1 is substituted with glutamine (5) The 35th tyrosine of SEQ ID NO: 1 is substituted with any of aspartic acid, glycine, lysine, leucine, asparagine, proline, serine, threonine, histidine (6) The 46th lysine of SEQ ID NO: 1 is substituted with isoleucine or threonine (7) The 48th glutamine of SEQ ID NO: 1 is substituted with histidine or leucine (8) The 50th alanine of SEQ ID NO: 1 is substituted with histidine (9) Sequence The 51st tyrosine of No. 1 is substituted with aspartic acid or histidine (10) The 54th glutamic acid of No. 1 is substituted with aspartic acid or glycine (11) The 56th asparagine of SEQ ID NO: 1 is substituted with threonine (12) The 59th glutamine of SEQ ID NO: 1 is substituted with arginine (13) SEQ ID NO: 1 Substitution of phenylalanine at position 61 with tyrosine (14) substitution of glutamic acid at position 64 of SEQ ID NO: 1 with aspartic acid (15) substitution of serine at position 65 of SEQ ID NO: 1 with arginine (16) position 71 of SEQ ID NO: 1 Alanine is substituted with aspartic acid (17) Phenylalanine at position 75 of SEQ ID NO: 1 is substituted with leucine, serine or tyrosine (18), aspartic acid at position 77 of SEQ ID NO: 1 is substituted with asparagine (19) SEQ ID NO: 1 Substitution of the 78 th alanine with serine to (20) the 82 th ass of SEQ ID NO: 1 Lagic acid is substituted with glutamic acid or valine (21) 90th glutamine of SEQ ID NO: 1 is substituted with arginine (22) Asparagine 92nd of SEQ ID NO: 1 is substituted with serine (23) Leucine 93 of SEQ ID NO: 1 is substituted Substitution with arginine or methionine (24) Substitution of the 95th threonine of SEQ ID NO: 1 with alanine or serine (25) Substitution of the 110th leucine of SEQ ID NO: 1 with glutamine (26) (27) tryptophan at position 116 of SEQ ID NO: 1 is substituted with leucine (28) phenylalanine at position 118 of SEQ ID NO: 1 is substituted with tyrosine (29) lysine at position 119 of SEQ ID NO: 1 is substituted with glutamic acid (30) The 120th glutamic acid of SEQ ID NO: 1 is substituted with valine (31) The 121st glutamic acid of No. 1 is substituted with aspartic acid or glycine (32) The 151st phenylalanine of SEQ ID NO: 1 is substituted with serine or tyrosine (33) The 155th serine of SEQ ID NO: 1 is substituted with threonine (34) The 163rd threonine of No. 1 substitutes for serine (35) The 167th serine of SEQ ID NO: 1 substitutes for glycine (36) The 169th serine of SEQ ID NO: 1 substitutes for glycine (37) 171st of SEQ ID NO: 1 Is substituted with tyrosine (38), and the asparagine at position 180 of SEQ ID NO: 1 is substituted with any of lysine, serine or isoleucine (39), and the threonine at position 185 of SEQ ID NO: 1 is substituted with serine (40). Glutamine at position 192 is substituted with lysine (B) Amino acid sequence described in SEQ ID NO: 1 Of the 17th to 192nd amino acid residues and at least the 35th tyrosine of SEQ ID NO: 1 in the 17th to 192nd amino acid residues is aspartic acid, glycine, lysine, leucine, asparagine, proline The Fc binding protein according to (A), which is substituted with any of serine, threonine and histidine.

  (C) At least the 35th tyrosine of SEQ ID NO: 1 is an asparagine, including the 17th to 192nd amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1, and at least the 35th tyrosine of SEQ ID NO: 1 in the 17th to 192nd amino acid residues Or the Fc binding protein according to (B), wherein proline is substituted.

  (D) SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 49 From amino acid sequence 33 to 208 The Fc binding protein according to (C), which comprises an amino acid residue of

  (E) SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 49, described in (D) Fc binding protein.

(F) The Fc binding protein according to any one of (A) to (C), wherein at least one of the following (41) to (44) has an amino acid substitution.
(41) leucine at position 66 of SEQ ID NO: 1 is substituted with histidine or arginine (42), glycine at position 147 of SEQ ID NO: 1 is substituted for aspartic acid (43), tyrosine at position 158 of SEQ ID NO: 1 is substituted for histidine (44) ) The adsorbent obtained by immobilizing the Fc binding protein according to any one of (G) (A) to (F) on an insoluble carrier, wherein the valine at position 176 of SEQ ID NO: 1 is substituted with phenylalanine.

  (H) A method for purifying an antibody using the adsorbent according to (G).

(I) A method of identifying differences in sugar chain structures possessed by antibodies by separating antibodies using the adsorbent described in (G).

  (J) An antibody obtained by the purification method described in (H).

  (K) A method for separating a sugar chain using the adsorbent according to (G).

  (L) A sugar chain obtained by the separation method described in (K).

(M) A polynucleotide encoding the Fc binding protein according to any of (A) to (F).

  (N) An expression vector comprising the polynucleotide according to (M).

  (O) A transformant obtained by transforming a host with the expression vector described in (N).

  (P) The transformant according to (O), wherein the host is E. coli.

(Q) Production of Fc binding protein, wherein Fc binding protein is expressed by culturing the transformant according to (O) or (P), and the Fc binding protein expressed from the culture is recovered Method.

  Hereinafter, the present invention will be described in detail.

  The Fc binding protein of the present invention is a protein having a binding property to the Fc region of an antibody, and at least among the extracellular region (region of EC of FIG. 1) of human FcγRIIIa consisting of the amino acid sequence of SEQ ID NO: 1 It is a protein comprising an amino acid residue from glycine 17 to glutamine 192, wherein an amino acid substitution at a specific position occurs in the amino acid residues 17 to 192. Therefore, the Fc binding protein of the present invention may contain all or part of the signal peptide region (S in FIG. 1) located N-terminal to the extracellular region, or the cell membrane located C-terminal to the extracellular region It may include all or part of the penetration region (TM in FIG. 1) and the extracellular region (C in FIG. 1). Specifically, the amino acid substitution at the specific position is Met18Arg (this notation represents that the methionine at position 18 of SEQ ID NO: 1 is substituted with arginine in the amino acid sequence described in SEQ ID NO: 1, etc. ), Val27Glu, Phe29Leu, Phe29Ser, Leu30Gln, Tyr35Asp, Tyr35Gly, Tyr35Lys, Tyr35Leu, Tyr35Leu, Tyr35Asn, Tyr35Pro, Tyr35Pro, Tyr35Ser, Tyr35His, Lys46Thr, Gln48His, Gln48Liph, a GiltyRiGil, a Gil, a Gil, a Gil Phe61Tyr, Glu64As Ser 65 Arg, Ala 71 Asp, Phe 75 Leu, Phe 75 Ser, Phe 75 Tyr, Asp 77 Asn, Ala 78 Ser, Asp 82 Glu, Asp 82 Val, Gln 90 Arg, Asn 92 Ser, Leu 93 Arg, , Ser155Thr, Thr163Ser, Ser167Gly, Ser169Gly, Phe171Tyr, Asn180Lys, Asn180Ser, Asn180Ile, Thr185Ser, Gln192 Of ys, it is one substitution of at least one. Among them, substitution of any one of Tyr35Asp, Tyr35Gly, Tyr35Lys, Tyr35Leu, Tyr35Asn, Tyr35Pro, Tyr35Pro, Tyr35Ser, Tyr35Thr, Tyr35His, or Glu121Gly is preferable because thermal stability is improved. In addition, wild type human FcγRIIIa is known to have a variant in which substitution of Leu66His, Leu66Arg, Gly147Asp, Tyr158His or Val176Phe has occurred, but these amino acid substitutions may be included in addition to the amino acid substitution at the specific position. .

  In addition, when producing Fc binding protein of this invention by performing amino acid substitution, about the amino acid residue of a specific position, as long as it has antibody binding activity, you may substitute by amino acids other than the amino acid mentioned above. An example is conservative substitution which substitutes between the amino acid in which physical property and / or chemical property of both amino acids are similar. It is known to those skilled in the art that conservative substitution is not limited to Fc binding proteins, but generally, the function of the protein is maintained between those in which substitution occurs and those in which substitution does not occur. Examples of conservative substitution include substitution occurring between glycine and alanine, between aspartic acid and glutamic acid, between serine and proline, or between glutamic acid and alanine (protein structure and function, Medical Science International, 9 , 2005).

In the Fc binding protein of the present invention, the number of substituted amino acids is not particularly limited. One example is the Fc binding protein shown in the following (a) to (h). These Fc binding proteins are preferred in terms of improving the stability to heat and acid.
(A) An amino acid substitution of Val27Glu and Tyr35Asn is generated at the 17th to 192nd amino acid residues including the 17th to 192nd amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1 Fc binding protein (Fc binding protein comprising the amino acid sequence from the 33rd to the 208th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 27)
(B) Amino acid substitution of Val27Glu, Tyr35Asn and Phe75Leu occurs at the 17th to 192nd amino acid residues including the 17th to 192nd amino acid residues of the amino acid sequence shown in SEQ ID NO: 1 Fc binding protein (Fc binding protein comprising the amino acid sequence from the 33rd to the 208th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 31)
(C) Amino acid substitution of Val27Glu, Tyr35Asn, Phe75Leu and Glu121Gly, which comprises the 17th to 192nd amino acid residues of the amino acid sequence shown in SEQ ID NO: 1 and the 17th to 192nd amino acid residues Fc binding protein (a Fc binding protein comprising an amino acid sequence from the 33rd to the 208th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 33)
(D) amino acid residues 17 to 192 in the amino acid sequence set forth in SEQ ID NO: 1 and at the amino acid residues 17 to 192, Val27Glu, Tyr35Asn, Phe75Leu, Asn92Ser and Glu121Gly Fc binding protein in which amino acid substitution has occurred (Fc binding protein comprising the amino acid sequence from the 33rd to the 208th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 37)
(E) At the amino acid residues 17 to 192 of the amino acid sequence set forth in SEQ ID NO: 1 and at the amino acid residues 17 to 192, Val27Glu, Tyr35Asn, Glu54Asp, Phe75Leu and Glu121Gly Fc binding protein in which amino acid substitution has occurred (Fc binding protein comprising amino acid sequence from the 33rd to the 208th amino acid sequence of the amino acid sequence described in SEQ ID NO: 41)
(F) Val27Glu, Tyr35Asn, Glu54Asp, Phe75Leu, Asn92Ser, and the like, which contain the 17th to 192nd amino acid residues of the amino acid sequence shown in SEQ ID NO: 1 and the 17th to 192nd amino acid residues. Fc-binding protein in which amino acid substitution of Glu121Gly is generated (Fc-binding protein including the amino acid sequence from the 33rd position to the 208th position of the amino acid sequence shown in SEQ ID NO: 43)
(G) Val27Glu, Tyr35Asn, Glu54Asp, Phe75Leu, Glu120Val, and the like, which contain the 17th to 192nd amino acid residues of the amino acid sequence shown in SEQ ID NO: 1 and the 17th to 192nd amino acid residues; Fc-binding protein in which amino acid substitution of Glu121Gly is generated (Fc-binding protein including the amino acid sequence from the 33rd position to the 208th position of the amino acid sequence shown in SEQ ID NO: 47)
(H) Val27Glu, Tyr35Asn, Glu54Asp, Phe75Leu, Asn92Ser, which comprises the 17th to 192nd amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1, and the 17th to 192nd amino acid residues; An Fc binding protein in which amino acid substitutions of Glu120Val and Glu121Gly occur (Fc binding protein comprising an amino acid sequence from the 33rd to the 208th amino acid sequence of the amino acid sequences shown in SEQ ID NO: 49).

  The Fc binding protein of the present invention may further have an oligopeptide useful for separation from a solution in the presence of a contaminant on the N-terminal side or C-terminal side. Examples of the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid and polyaspartic acid. In addition, a cysteine-containing oligopeptide useful for immobilizing the Fc-binding protein of the present invention on a solid phase such as a support for chromatography is the N-terminal side or C-terminal side of the Fc-binding protein of the present invention It may be added to The length of the oligopeptide added to the N-terminal side or C-terminal side of the Fc binding protein is not particularly limited as long as it does not impair the IgG binding property or stability of the Fc binding protein of the present invention. When adding the oligopeptide to the Fc binding protein of the present invention, after producing a polynucleotide encoding the oligopeptide, the N-terminal side of the Fc binding protein is genetically engineered using a method well known to those skilled in the art Alternatively, they may be added to the C-terminal side, or the chemically synthesized oligopeptide may be added by chemically binding to the N-terminal side or C-terminal side of the Fc binding protein of the present invention. Furthermore, to the N-terminal side of the Fc binding protein of the present invention, a signal peptide may be added to promote efficient expression in the host. Examples of the signal peptide when the host is E. coli include secreting proteins in the periplasm such as PelB, DsbA, MalE (region from the 1st to the 26th of the amino acid sequences described in UniProt No. P0AEX9), TorT, etc. A signal peptide can be illustrated (Unexamined-Japanese-Patent No. 2011-097898).

As an example of the method for producing the polynucleotide of the present invention,
(I) A method of artificially synthesizing a polynucleotide comprising the nucleotide sequence by converting the amino acid sequence of the Fc binding protein of the present invention into a nucleotide sequence,
(II) A polynucleotide comprising an entire or partial sequence of Fc binding protein is prepared directly or artificially, or is prepared from the cDNA of Fc binding protein or the like using a DNA amplification method such as PCR, and the polynucleotide is appropriately prepared Can be exemplified.
In the method (I), when converting an amino acid sequence to a nucleotide sequence, it is preferable to convert in consideration of the usage frequency of codons in the host to be transformed. As an example, when the host is Escherichia coli, AGA / AGG / CGG / CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly) However, since CCC in Proline (Pro) is used less frequently (because it is a so-called rare codon), conversion may be performed so as to avoid those codons. Analysis of codon usage can also be performed by using a public database (eg, Codon Usage Database on the homepage of Kazusa DNA Research Institute).

When introducing a mutation into the polynucleotide of the present invention, error-prone PCR can be used. The reaction conditions in the error-prone PCR method are not particularly limited as long as they can introduce a desired mutation into a polynucleotide encoding human FcγRI (or Fc binding protein), for example, four deoxynucleotides as substrates ( Polynucleotides are prepared by making the concentration of dATP / dTTP / dCTP / dGTP) heterogeneous and adding MnCl ₂ to the PCR reaction solution at a concentration of 0.01 to 10 mM (preferably 0.1 to 1 mM) to conduct PCR. Mutations can be introduced into Further, as a mutation introduction method other than the error-prone PCR method, a polynucleotide containing the whole or a partial sequence of human FcγRI is brought into contact with or reacted with an agent serving as a mutagen, or irradiated with ultraviolet light to mutate the polynucleotide. The method to introduce and produce is mentioned. As the drug to be used as a mutagen in the method, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, hydrazine and the like, mutagenic agents commonly used by those skilled in the art may be used. .

  When a host is transformed with the polynucleotide of the present invention, the polynucleotide of the present invention may be used, but an expression vector (eg, bacteriophage, cosmid or the like usually used for transformation of prokaryotic cells or eukaryotic cells) may be used. It is more preferable to use one in which the polynucleotide of the present invention is inserted at an appropriate position of a plasmid etc.). The expression vector is not particularly limited as long as it stably exists in the host to be transformed and can be replicated, and when using E. coli as the host, pET plasmid vector, pUC plasmid vector, pTrc plasmid vector, pCDF plasmid vector , PBBR plasmid vectors can be exemplified. The appropriate position means a position which does not destroy the replication function of the expression vector, the desired antibiotic marker, and the region involved in transferability. When inserting the polynucleotide of the present invention into the expression vector, it is preferable to insert in a state of being linked to a functional polynucleotide such as a promoter necessary for expression. Examples of such a promoter include trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, λ phage promoter, λ PR promoter, λ PR promoter and the like when the host is E. coli.

  In order to transform a host using an expression vector (hereinafter referred to as the expression vector of the present invention) into which the polynucleotide of the present invention prepared by the above-mentioned method is inserted, the transformation may be carried out by a method commonly used by those skilled in the art. For example, when selecting a microorganism belonging to the genus Escherichia (E. coli JM109 strain, E. coli BL21 (DE3) strain, E. coli W3110 strain, etc.) as a host, known documents (eg Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992) The transformation may be carried out by the method described in 1.). The transformant obtained by transformation according to the above-mentioned method is screened by an appropriate method to obtain a transformant capable of expressing the Fc binding protein of the present invention (hereinafter referred to as the transformant of the present invention) ) Can be obtained. The host expressing the Fc binding protein of the present invention is not particularly limited. For example, animal cells (CHO cells, HEK cells, Hela cells, COS cells, etc.), yeasts (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha) Schizosaccharomyces japonicus, Schizosaccharomyces octosporus, Schizosaccharomyces pombe etc., insect cells (Sf9, Sf21 etc.), E. coli (JM109 strain, BL21 (DE3) strain, W3110 strain etc.) and Bacillus subtilis. It is preferable to use animal cells and E. coli as a host in terms of productivity, and it is more preferable to use E. coli as a host.

  In order to prepare the expression vector of the present invention from the transformant of the present invention, the alkaline extract method or a commercially available product such as QIAprep Spin Miniprep kit (manufactured by Qiagen) from the culture obtained by culturing the transformant of the present invention It may be prepared using an extraction kit of The Fc binding protein of the present invention can be produced by culturing the transformant of the present invention and recovering the Fc binding protein of the present invention from the obtained culture. In the present specification, the term "culture" includes not only cultured cells of the transformant of the present invention but also the medium used for culture. The transformant used in the protein production method of the present invention may be cultured in a medium suitable for culturing the target host, and when the host is E. coli, LB (Luria-Bertani) medium supplemented with a necessary nutrient source is preferable An example of the culture medium is given. In order to selectively grow the transformant of the present invention depending on the presence or absence of the introduction of the vector of the present invention, it is preferable to culture the medium with a drug corresponding to the drug resistance gene contained in the vector. For example, when the vector contains a kanamycin resistance gene, kanamycin may be added to the medium. In addition to carbon, nitrogen and inorganic salt sources, the medium may also contain suitable nutrient sources, optionally selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate and dithiothreitol. May contain one or more reducing agents. Furthermore, a reagent that promotes protein secretion from the transformant to the culture solution, such as glycine, may be added. Specifically, when the host is E. coli, the concentration of glycine is 2% (w / v) or less of the culture medium. It is preferable to add it. The culture temperature is generally 10 ° C. to 40 ° C., preferably 20 ° C. to 37 ° C., more preferably around 25 ° C. when the host is E. coli, and may be selected according to the characteristics of the protein to be expressed. The pH of the medium is pH 6.8 to pH 7.4, preferably around pH 7.0, when the host is E. coli. When the vector of the present invention contains an inducible promoter, induction is preferably performed under conditions such that the Fc binding protein of the present invention can be expressed well. As an inducer, IPTG (isopropyl- (beta) -D-thiogalactopyranoside) can be illustrated. When the host is E. coli, the turbidity of the culture solution (absorbance at 600 nm) is measured, and when it reaches about 0.5 to 1.0, an appropriate amount of IPTG is added, followed by culturing to obtain Fc binding. Protein expression can be induced. The addition concentration of IPTG may be appropriately selected from the range of 0.005 to 1.0 mM, preferably in the range of 0.01 to 0.5 mM. Various conditions for IPTG induction may be performed under conditions well known in the art.

  In order to recover the Fc-binding protein of the present invention from the culture obtained by culturing the transformant of the present invention, a method suitable for the expression form of the Fc-binding protein of the present invention in the transformant of the present invention The Fc-binding protein of the present invention may be recovered by separation / purification from the culture. For example, when expressing in the culture supernatant, the cells may be separated by centrifugation, and the Fc binding protein of the present invention may be purified from the obtained culture supernatant. In addition, when it is expressed in cells (including periplasm), after collecting the cells by centrifugation, the cells are disrupted by adding an enzyme treatment agent, a surfactant and the like to obtain Fc of the present invention. The binding protein may be extracted and then purified. In order to purify the Fc binding protein of the present invention, methods known in the art may be used, such as separation / purification using liquid chromatography as an example. Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc. The Fc binding ability of the present invention can be achieved by performing a purification operation by combining these chromatographys. Proteins can be prepared to high purity. As a method of measuring the binding activity of the obtained Fc binding protein of the present invention to IgG, for example, the binding activity to IgG is determined using Enzyme-Linked Immunosorbent Assay (hereinafter referred to as ELISA) method, surface plasmon resonance method or the like. It should be measured. As IgG used for measurement of binding activity, human IgG is preferable, and human IgG1 and human IgG3 are particularly preferable.

  The adsorbent of the present invention can be produced by binding the Fc binding protein of the present invention to an insoluble carrier. The insoluble carrier is not particularly limited, and carriers made of polysaccharides such as agarose, alginate (alginate), carrageenan, chitin, cellulose, dextrin, dextran and starch as a raw material, polyvinyl alcohol, polymethacrylate, poly (2- Examples of such carriers include carriers made of synthetic polymers such as hydroxyethyl methacrylate) and polyurethane, and carriers made of ceramics such as silica. Among them, carriers derived from polysaccharides and carriers derived from synthetic polymers are preferred as insoluble carriers. Examples of the preferred carrier include polymethacrylate gel into which a hydroxyl group is introduced such as Toyopearl (manufactured by Tosoh Corp.), agarose gel such as Sepharose (manufactured by GE Healthcare), and cellulose gel such as Celfine (manufactured by JNC). . There is no particular limitation on the form of the insoluble carrier, and it may be particulate or non-particulate, porous or non-porous.

  In order to immobilize the Fc binding protein on an insoluble carrier, N-hydroxysuccinimide (NHS) activated ester group, epoxy group, carboxyl group, maleimide group, haloacetyl group, tresyl group, formyl group haloacetamide is used as the insoluble carrier And the like, and immobilized by covalently bonding a human Fc-binding protein and an insoluble carrier via the active group. The carrier to which the active group is attached may be a commercially available carrier as it is, or may be prepared by introducing an active group onto the surface of the carrier under appropriate reaction conditions. Commercially available carriers to which an active group has been added include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M (all manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4 Fast Flow, Epoxy-activated Sepharose 6B (all are manufactured by GE Healthcare) and SulfoLink Coupling Resin (manufactured by Thermo Scientific).

  On the other hand, as a method of introducing an active group on the surface of a carrier, a method of reacting one of a compound having two or more active sites with a hydroxyl group, an epoxy group, a carboxyl group, an amino group, etc. present on the carrier surface is exemplified it can. Among the examples of the compound, epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether can be exemplified as a compound for introducing an epoxy group to a hydroxyl group or amino group on the carrier surface. As a compound which introduce | transduces a carboxyl group into the support | carrier surface, after introduce | transducing an epoxy group into the support | carrier surface with the said compound, 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3- Aminopropionic acid, 4-aminobutyric acid, 6-aminohexanoic acid can be exemplified.

  As a compound for introducing a maleimide group to a hydroxyl group, an epoxy group, a carboxyl group or an amino group present on the carrier surface, N- (ε-maleimidocaproic acid) hydrazide, N- (ε-maleimidopropionic acid) hydrazide, 4- [4 4-N-maleimidophenyl] acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1- (4-aminophenyl) maleimide, 1- (3-aminophenyl) maleimide, 4- (Maleimido) phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, (N- [α-maleimidoacetoxy] succinimide ester), (m-maleimidobenzoyl) N-hydroxysuccinimide ester, Succinimidyl-4- [maleimidomethyl] cyclohexane-1-carbonyl- [6-aminohexanoic acid], (succinimidyl-4- [maleimidomethyl] cyclohexane-1-carboxylic acid), (p-maleimidobenzoyl) N-hydroxysuccinimide An ester, (m-maleimido benzoyl) N-hydroxy succinimide ester can be illustrated.

  Examples of compounds for introducing a haloacetyl group to a hydroxyl group or amino group present on the surface of a carrier include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, Iodoacetic anhydride, 2- (iodoacetamido) acetic acid-N-hydroxysuccinimide ester, 3- (bromoacetamido) propionic acid-N-hydroxysuccinimide ester, 4- (iodoacetyl) aminobenzoic acid-N-hydroxysuccinimide ester It can be illustrated. In addition, after making (omega) -alkenyl alkane glycidyl ether react with the hydroxyl group and amino group which exist on the support | carrier surface, the method of halogenating and activating (omega) -alkenyl part with a halogenating agent can also be illustrated. Examples of ω-alkenyl alkane glycidyl ether include allyl glycidyl ether, 3-butenyl glycidyl ether and 4-pentenyl glycidyl ether, and examples of a halogenating agent include N-chlorosuccinimide, N-bromosuccinimide and N-iodosuccinimide it can.

  As another example of the method of introducing an active group to the surface of a carrier, there is a method of introducing an activating group to a carboxyl group present on the surface of a carrier using a condensing agent and an additive. As a condensing agent, 1-ethyl 3- (3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexyl carbodiimide, carbonyl diimidazole can be illustrated. Moreover, N-hydroxy succinimide (NHS), 4-nitrophenol, 1-hydroxy benztriazole can be illustrated as an additive.

  As a buffer used when immobilizing the Fc binding protein of the present invention on an insoluble carrier, acetate buffer, phosphate buffer, MES (2-Morpholinoethanesulfonic acid) buffer, HEPES (2- [4- (2- (2-) Examples thereof include Hydroxyethyl) -1-piperidinyl] ethanesulfonic acid) buffer, Tris buffer, and borate buffer. The reaction temperature for immobilization may be appropriately set in consideration of the reactivity of the active group and the stability of the Fc binding protein of the present invention from the temperature range of 5 ° C. to 50 ° C., preferably It is in the range of 10 ° C to 35 ° C.

  In order to purify an antibody having a sugar chain using the adsorbent of the present invention obtained by immobilizing the Fc binding protein of the present invention on an insoluble carrier, for example, a sugar packed in a column packed with the adsorbent of the present invention After the antibody having a sugar chain is specifically adsorbed to the adsorbent of the present invention by adding a buffer solution containing an antibody having a chain using a pump or other delivery means, an appropriate elution solution is prepared. The antibody having a sugar chain may be eluted by adding to the column. The antibody having a sugar chain that can be purified by the adsorbent of the present invention may be an antibody having at least an Fc region of an antibody having a sugar chain, which has an affinity to the Fc binding protein. As an example, a chimeric antibody, a humanized antibody, a human antibody or an amino acid substitution product thereof generally used as an antibody used for antibody medicine can be mentioned. In addition, bispecific antibody (bispecific antibody), fusion antibody of Fc region of antibody with sugar chain and other protein, complex of antibody with Fc region of sugar chain and drug (ADC), etc. Even artificially altered antibodies can be purified with the adsorbent of the present invention. In addition, it is preferable to equilibrate the column using an appropriate buffer before adding a buffer containing an antibody having a sugar chain to the column, since the antibody having a sugar chain can be purified with higher purity. Examples of the buffer solution include phosphate buffer solutions and buffers containing an inorganic salt as a component, and the pH of the buffer solution is pH 3 to 10, preferably pH 5 to 8.

  In order to elute an antibody having a sugar chain adsorbed to the adsorbent of the present invention, it is sufficient to weaken the interaction between the antibody having a sugar chain and the ligand (the Fc binding protein of the present invention), specifically In the above, pH change by buffer solution, counter peptide, temperature change, salt concentration change can be exemplified. As a specific example of an elution solution for eluting an antibody having a sugar chain adsorbed to the adsorbent of the present invention, it is more acidic than a solution used for adsorbing an antibody having a sugar chain to the adsorbent of the present invention The side buffer solution is raised. As a type of buffer, a citrate buffer having a buffer capacity on the acid side, a glycine hydrochloride buffer, and an acetate buffer can be exemplified. The pH of the buffer may be set within the range that does not impair the function of the antibody, preferably 2.5 to 6.0, more preferably 3.0 to 5.0, and still more preferably 3.3 to 4. It is 0.

  When purifying the antibody from the solution containing the antibody having a sugar chain using the adsorbent of the present invention, the elution position (elution fraction) of the antibody differs depending on the difference in the sugar chain structure of the antibody. Therefore, by separating the antibody using the adsorbent of the present invention, it is possible to identify the difference in sugar chain structure possessed by the antibody. There is no particular limitation on the structure of distinguishable sugar chains, and as an example, a sugar chain added when an antibody is expressed using as a host a cell derived from an animal such as CHO cells or a yeast such as Pichia yeast or Saccharomyces yeast, Examples include sugar chains possessed by antibodies, and sugar chains added to antibodies by chemical synthesis. Moreover, since the adsorbent of the present invention can be separated based on the difference in sugar chain structure possessed by the antibody, it can also be used for separation of sugar chains themselves.

  The Fc binding protein of the present invention is a protein in which an amino acid residue at a specific position in the extracellular region of human FcγRIIIa is replaced with another amino acid residue. The Fc binding protein of the present invention has improved heat and acid stability as compared to wild type human FcγRIIIa. Therefore, the Fc binding protein of the present invention is useful as a ligand of an adsorbent for separating immunoglobulins.

FIG. 1 is a schematic view of human FcγRIIIa. The numbers in the figure indicate the numbers of the amino acid sequences set forth in SEQ ID NO: 1. In the figure, S indicates a signal sequence, EC indicates an extracellular region, TM indicates a cell transmembrane region, and C indicates an intracellular region. It is a figure which shows the result of having evaluated the antibody binding activity of Fc binding protein which carried out 1 amino acid substitution. The wild type in the figure indicates Fc binding protein without amino acid substitution. It is a figure which shows the result of having evaluated the thermostability of Fc binding protein which carried out 1 amino acid substitution. The wild type in the figure indicates Fc binding protein without amino acid substitution. It is the chromatograph chart which showed elution of the antibody using FcR5a fixed gel. Fr in the figure indicates the position of the collected fraction. It is the figure which showed the list of the sugar_chain | carbohydrate structure added to the antibody. N1 to N8 in the figure correspond to N1 to N8 in Table 10, and M1 and M2 correspond to M1 and M2 in Table 11. It is a figure (SDS-PAGE) which showed the result of antibody purification using FcR5a fixed gel. (A) shows the result of purification of human IgG1 and (B) shows the result of purification of human IgG3.

  Hereinafter, examples are shown to describe the present invention in more detail, but the present invention is not limited to the examples.

Example 1 Preparation of Fc-Binding Protein Expression Vector (1) Based on the amino acid sequence from 17th glycine (Gly) to 192nd glutamine (Gln) of the human FcγRIIIa amino acid sequence described in SEQ ID NO: 1 Using DNAworks method (Nucleic Acids Res., 30, e 43, 2002), nucleotide sequences in which codons were converted from human to E. coli were designed. The designed nucleotide sequence is shown in SEQ ID NO: 2.
(2) In order to produce a polynucleotide comprising the sequence set forth in SEQ ID NO: 2, an oligonucleotide consisting of the sequence set forth in SEQ ID NOs: 3 to 20 is synthesized, and two-step PCR shown below is carried out using said oligonucleotide. Did.
(2-1) The first-step PCR prepares a reaction solution having the composition shown in Table 1, heat-treats the reaction solution at 98 ° C. for 5 minutes, and then performs the first step at 98 ° C. for 10 seconds, 5 at 62 ° C. A polynucleotide was synthesized by repeating the cycle of the second step of 2 seconds, and the reaction of the third step of 90 seconds at 72 ° C. as one cycle, to obtain FcRp1. The DNA mix in Table 1 means a solution obtained by sampling and mixing a predetermined amount of 18 kinds of oligonucleotides consisting of the sequences described in SEQ ID NOs: 3 to 20, respectively.

（２−２）二段階目のＰＣＲは、（２−１）で合成したＦｃＲｐ１を鋳型とし、配列番号２１（５’−ＴＡＧＣＣＡＴＧＧＧＣＡＴＧＣＧＴＡＣＣＧＡＡＧＡＴＣＴＧＣＣＧＡＡＡＧＣ−３’）および配列番号２２（５’−ＣＣＣＡＡＧＣＴＴＡＡＴＧＡＴＧＡＴＧＡＴＧＡＴＧＡＴＧＧＣＣＣＣＣＴＴＧＧＧＴＡＡＴＧＧＴＡＡＴＡＴＴＣＡＣＧＧＴＣＴＣＧＣＴＧＣ−３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして実施した。具体的には、表２に示す組成の反応液を調製し、当該反応液を９８℃で５分熱処理後、９８℃で１０秒間の第１ステップ、６２℃で５秒間の第２ステップ、７２℃で１．５分間の第３ステップを１サイクルとする反応を３０サイクル繰り返すことで実施した。

(2-2) The second step PCR uses FcRp1 synthesized in (2-1) as a template and SEQ ID NO: 21 (5'- TAGCCATGGGCATGCGTACCGAAGATCTGCCGAAAGC-3 ') and SEQ ID NO: 22 (5'- CCCAAGCTTAATATGATGATGATGGCCCCTTGGGTAATGGTAATATTCACGGTCTCGCTGCTGC-3) An oligonucleotide consisting of the sequence described in was carried out as a PCR primer. Specifically, a reaction liquid having a composition shown in Table 2 is prepared, and the reaction liquid is heat-treated at 98 ° C. for 5 minutes and then subjected to a first step at 98 ° C. for 10 seconds, a second step at 62 ° C. for 5 seconds, 72 The reaction was carried out by repeating 30 cycles of the reaction consisting of the third step at 1.5 ° C. and one cycle.

（３）（２）で得られたポリヌクレオチドを精製し、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化後、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１−２０６０４６号公報）にライゲーションし、当該ライゲーション産物を用いて大腸菌ＢＬ２１株（ＤＥ３）を形質転換した。
（４）得られた形質転換体を５０μｇ／ｍＬのカナマイシンンを含むＬＢ培地にて培養後、ＱＩＡｐｒｅｐ Ｓｐｉｎ Ｍｉｎｉｐｒｅｐ ｋｉｔ（キアゲン社製）を用いて、発現ベクターｐＥＴ−ｅＦｃＲを抽出した。
（５）（４）で作製した発現ベクターｐＥＴ−ｅＦｃＲのうち、ヒトＦｃγＲＩＩＩａをコードするポリヌクレオチドおよびその周辺の領域について、チェーンターミネータ法に基づくＢｉｇ Ｄｙｅ Ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ ｒｅａｄｙ Ｒｅａｃｔｉｏｎ ｋｉｔ（ライフサイエンス社製）を用いてサイクルシークエンス反応に供し、全自動ＤＮＡシークエンサーＡＢＩ Ｐｒｉｓｍ ３７００ ＤＮＡ ａｎａｌｙｚｅｒ（ライフサイエンス社製）にてヌクレオチド配列を解析した。なお当該解析の際、配列番号２３（５’−ＴＡＡＴＡＣＧＡＣＴＣＡＣＴＡＴＡＧＧＧ−３’）または配列番号２４（５’−ＴＡＴＧＣＴＡＧＴＴＡＴＴＧＣＴＣＡＧ−３’）に記載の配列からなるオリゴヌクレオチドをシークエンス用プライマーとして使用した。

(3) The polynucleotide obtained in (2) is purified, digested with restriction enzymes NcoI and HindIII, and then ligated in expression vector pETMalE (Japanese Patent Laid-Open No. 2011-206046) which has been previously digested with restriction enzymes NcoI and HindIII, The ligation product was used to transform E. coli strain BL21 (DE3).
(4) The obtained transformant was cultured in LB medium containing 50 μg / mL kanamycin, and then the expression vector pET-eFcR was extracted using QIAprep Spin Miniprep kit (manufactured by Qiagen).
(5) Of the expression vector pET-eFcR prepared in (4), for the polynucleotide encoding human FcγRIIIa and the surrounding region, Big Dye Terminator Cycle Sequencing ready Reaction kit (manufactured by Life Science) based on the chain terminator method And subjected to a cycle sequencing reaction, and the nucleotide sequence was analyzed by a fully automatic DNA sequencer ABI Prism 3700 DNA analyzer (manufactured by Life Science). In the analysis, an oligonucleotide consisting of the sequence described in SEQ ID NO: 23 (5′-TAATACGACTCACTATAGGG-3 ′) or SEQ ID NO: 24 (5′-TATGCTAGTTATTGCTCAG-3 ′) was used as a primer for sequencing.

  The amino acid sequence of the polypeptide expressed by the expression vector pET-eFcR is shown in SEQ ID NO: 25 and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 26. In SEQ ID NO: 25, the 1st methionine (Met) to the 26th alanine (Ala) is a MalE signal peptide, the 27th lysine (Lys) to the 32nd methionine (Met) is a linker sequence, The 33rd glycine (Gly) to the 208th glutamine (Gln) is the extracellular region of human FcγRIIIa (the 17th to 192th regions of SEQ ID NO: 1), and the 209th to 210th glycines (Gly) Gly) is a linker sequence, and histidine (His) at positions 211 to 216 is a tag sequence.

Example 2 Mutation Introduction to Fc-Binding Protein and Preparation of Library Of the Fc-binding protein expression vector pER-eFcR prepared in Example 1, the polynucleotide part encoding the Fc-binding protein was subjected to error-prone PCR by PCR. Mutations were randomly introduced.
(1) An error-prone PCR was performed using pET-eFcR prepared in Example 1 as a template. After preparing a reaction solution having the composition shown in Table 3, the error-prone PCR heat-processes the reaction solution at 95 ° C. for 2 minutes, and the first step for 30 seconds at 95 ° C., the second step for 30 seconds at 60 ° C. 72 The reaction was carried out for 35 cycles with one cycle of the third step of 90 ° C. for 90 seconds, and finally the heat treatment was carried out for 7 minutes at 72 ° C. The error-prone PCR successfully introduced a mutation into the polynucleotide encoding the Fc binding protein, and the average mutation introduction rate was 1.26%.

（２）（１）で得られたＰＣＲ産物を精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ同制限酵素で消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１−２０６０４６号公報）にライゲーションした。
（３）ライゲーション反応終了後、反応液をエレクトロポレーション法により大腸菌ＢＬ２１（ＤＥ３）株に導入し、５０μｇ／ｍＬのカナマイシンを含むＬＢプレート培地で培養（３７℃で１８時間）後、プレート上に形成したコロニーをランダム変異体ライブラリーとした。

(2) After purifying the PCR product obtained in (1), it was digested with restriction enzymes NcoI and HindIII, and ligated to an expression vector pETMalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with the restriction enzymes.
(3) After completion of the ligation reaction, the reaction solution is introduced into E. coli BL21 (DE3) strain by electroporation and cultured on LB plate medium containing 50 μg / mL kanamycin (18 hours at 37 ° C.), and then plated on the plate The formed colonies were used as a random mutant library.

Example 3 Screening of heat-stabilized Fc binding protein (1) 2YT liquid medium (peptone 16 g / L, yeast containing 50 μg / mL kanamycin) of the random mutant library (transformant) prepared in Example 2 The extract was inoculated in 200 μL of 10 g / L of extract, 5 g / L of sodium chloride, and cultured with shaking overnight at 30 ° C. using a 96-well deep well plate.
(2) After culture, 5 μL of the culture broth was transferred to 500 μL of 0.05 mM IPTG (isopropyl-β-D-thiogalactopyranoside), 0.3% glycine and 50 μg / mL kanamycin in 2YT liquid medium, and 96 Using a well deep well plate, shake culture was further performed at 20 ° C. overnight.
(3) After culture, the culture supernatant obtained by centrifugation was diluted twice with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride. The diluted solution was heat treated at 45 ° C. for 10 minutes.
The ELISA method which shows the antibody binding activity of Fc binding protein when heat treatment of (4) and (3) and the antibody binding activity of Fc binding protein when heat treatment of (3) is not performed is shown below, respectively. The residual activity was calculated by dividing the antibody binding activity of the Fc binding protein when heat treatment was performed by the antibody binding activity of the Fc binding protein when heat treatment was not performed.
(4-1) Immobilize human globulin gamma globulin preparation (manufactured by Chemo- and Serum Therapy Research Institute) at 1 μg / well in wells of 96-well microplate (18 hours at 4 ° C), and after immobilization, 2 Blocking was carried out with 20 mM Tris-HCl buffer (pH 7.4) containing% (w / v) of SKIM MILK (manufactured by BD) and 150 mM of sodium chloride.
(4-2) Fc for evaluating antibody binding activity after washing with washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% [w / v] Tween 20, 150 mM NaCl) A solution containing binding protein was added to react Fc binding protein with immobilized gamma globulin (1 hour at 30 ° C.).
(4-3) After completion of the reaction, the plate was washed with the above-mentioned washing buffer, and an Anti-6His antibody (manufactured by Bethyl Laboratories) diluted to 100 ng / mL was added at 100 μL / well.
(4-4) After reacting at 30 ° C. for 1 hour and washing with the above-mentioned washing buffer, TMB Peroxidase Substrate (manufactured by KPL) was added at 50 μL / well. Color development was stopped by adding 1 M phosphoric acid at 50 μL / well, and absorbance at 450 nm was measured with a microplate reader (manufactured by TEKAN).
(5) Evaluate about 2700 transformants by the method of (4), and among them, an Fc binding protein having improved thermal stability as compared to a wild type (without amino acid substitution) Fc binding protein The transformant to express was selected. The selected transformant was cultured, and an expression vector was prepared using QIAprep Spin Miniprep kit (manufactured by Qiagen).
(6) The nucleotide sequence of the polynucleotide region encoding the Fc binding protein inserted into the obtained expression vector is analyzed by the same method as described in Example 1 (5) to identify the amino acid mutation site did.

  Table 4 summarizes the position of amino acid substitution relative to wild-type (without amino acid substitution) Fc binding protein of the Fc binding protein expressed by the transformant selected in (5) and the residual activity (%) after heat treatment Shown in. In the amino acid sequence set forth in SEQ ID NO: 1, Met18Arg (this notation is SEQ ID NO: 1), which comprises the amino acid residue from glycine 17 to glutamine 192 in the amino acid residue 17 and the amino acid residue 17 to 192 The same also applies to the fact that the 18th methionine of 1 is substituted with arginine), Val27Glu, Phe29Leu, Phe29Ser, Leu30Gln, Tyr35Asn, Tyr35Asn, Tyr35Asp, Tyr35Ser, Tyr35His, Lys46Ile, Lys46Thr, Gln48His, Gln48Leu, Ala50His, Tyr51His, Tyr51His, , Glu54Asp, Glu54Gly, Asn56Thr, Gln59Arg, Phe61Tyr, Glu64Asp, Ser6 Arg, Ala 71 Asp, Phe 75 Leu, Phe 75 Tyr, Phe 75 Tyr, Asp 77 Asn, Ala 78 Ser, Asp 82 Glu, Asp 82 Val, Gln 90 Arg, Asn 92 Ser, Leu 93 Arg, Leu 93 Arg, All of Ser155Thr, Thr163Ser, Ser167Gly, Ser169Gly, Phe171Tyr, Asn180Lys, Asn180Ser, Asn180Ile, Thr185Ser, Gln192Lys Fc binding proteins Kano amino acid substitution is at least one occurs, it can be said that the thermal stability as compared to wild-type Fc binding protein is improved.

表４に示す、アミノ酸置換されたＦｃ結合性タンパク質のうち、最も残存活性の高い、Ｖａｌ２７ＧｌｕおよびＴｙｒ３５Ａｓｎのアミノ酸置換が生じたＦｃ結合性タンパク質をＦｃＲ２と命名し、ＦｃＲ２をコードするポリヌクレオチドを含む発現ベクターをｐＥＴ−ＦｃＲ２と命名した。ＦｃＲ２のアミノ酸配列を配列番号２７に、ＦｃＲ２をコードするポリヌクレオチドの配列を配列番号２８に示す。なお配列番号２７において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ２のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号２７において、Ｖａｌ２７Ｇｌｕのグルタミン酸は４３番目、Ｔｙｒ３５Ａｓｎのアスパラギンは５１番目の位置にそれぞれ存在する。

Among the amino acid substituted Fc binding proteins shown in Table 4, an Fc binding protein in which amino acid substitution of Val27Glu and Tyr35Asn is generated with the highest residual activity is designated as FcR2 and expression including a polynucleotide encoding FcR2 The vector was named pET-FcR2. The amino acid sequence of FcR2 is shown in SEQ ID NO: 27 and the sequence of a polynucleotide encoding FcR2 is shown in SEQ ID NO: 28. In SEQ ID NO: 27, the 1st methionine (Met) to the 26th alanine (Ala) is a MalE signal peptide, the 27th lysine (Lys) to the 32nd methionine (Met) is a linker sequence, The 33rd glycine (Gly) to the 208th glutamine (Gln) is the amino acid sequence of FcR2 (corresponding to the 17th to 192th regions of SEQ ID NO: 1), the 209th to 210th glycine (Gly) Is a linker sequence, and histidine (His) at positions 211 to 216 is a tag sequence. In SEQ ID NO: 27, the glutamate at Val27Glu is at position 43, and the asparagine at Tyr35Asn is at position 51.

Example 4 Preparation of Amino Acid-Substituted Fc-Binding Protein The stability was further improved by accumulating the amino acid substitutions involved in the improvement of the thermal stability of the Fc-binding protein as found in Example 3. The accumulation of substituted amino acids was mainly performed using PCR, and seven kinds of Fc binding proteins shown in (a) to (g) below were produced.
(A) FcR3 in which amino acid substitution of Phe75Leu was further performed on FcR2
(B) FcR4 further subjected to amino acid substitution of Phe75Leu and Glu121Gly for FcR2
(C) FcR5a wherein amino acid substitution of Asn92Ser was further performed on FcR4
(D) FcR5b in which amino acid substitution of Glu54Asp was further performed on FcR4
(E) FcR6a in which amino acid substitution of Glu54Asp was further performed on FcR5a
(F) FcR6b in which amino acid substitution of Glu120Val was further performed on FcR5b
(G) FcR7 in which amino acid substitution of Glu120Val was further performed on FcR6a
Hereinafter, methods for producing each Fc binding protein will be described in detail.

(A) FcR3
Among the amino acid substitutions involved in the thermal stability improvement that were revealed in Example 3, Val27Glu, Tyr35Asn and Phe75Leu were selected, and FcR3 in which those substitutions were accumulated in a wild-type Fc binding protein was prepared. Specifically, FcR3 was produced by performing mutagenesis to generate Phe75Leu to a polynucleotide encoding FcR2.
(A-1) PCR was performed using pET-FcR2 obtained in Example 3 as a template. The primer in the said PCR used the oligonucleotide which consists of a sequence as described in sequence number 24 and sequence number 29 (5'-AGCCAGGCGAGCAGCTACCTTATTGATGCG-3 '). After preparing the reaction solution having the composition shown in Table 5, the PCR is heat-treated at 98 ° C for 5 minutes, and the first step at 98 ° C for 10 seconds, the second step at 55 ° C for 5 seconds, 72 ° C. The reaction was carried out for 30 cycles, in which the third step of 1 minute was one cycle, and finally the heat treatment was carried out at 72 ° C. for 7 minutes. The amplified PCR product was subjected to agarose gel electrophoresis, and the gel was purified using QIAquick Gel Extraction kit (manufactured by Qiagen). The purified PCR product was called m3F.

（ａ−２）実施例３で取得した、ｐＥＴ−ＦｃＲ２を鋳型とし、配列番号２３および配列番号３０（５’−ＣＣＡＣＣＧＴＣＧＣＣＧＣＡＴＣＡＡＴＡＡＧＧＴＡＧＣＴＧＣ−３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ａ−１）と同様に行なった。精製したＰＣＲ産物をｍ３Ｒとした。
（ａ−３）（ａ−１）および（ａ−２）で得られた２種類のＰＣＲ産物（ｍ３Ｆ、ｍ３Ｒ）を混合し、表６に示す組成の反応液を調製した。当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を５サイクル行なうＰＣＲを行ない、ｍ３Ｆとｍ３Ｒを連結したＰＣＲ産物ｍ３ｐを得た。

(A-2) Using pET-FcR2 obtained in Example 3 as a template and the oligonucleotide consisting of the sequence described in SEQ ID NO: 23 and SEQ ID NO: 30 (5′-CCACCGTCGCCGGCATCAATAAGGTAGCTGC-3 ′) as PCR primers , (A-1). The purified PCR product was called m3R.
(A-3) The two PCR products (m3F, m3R) obtained in (a-1) and (a-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, then the first step of 98 ° C. for 10 seconds, the second step of 55 ° C. for 5 seconds, the reaction of 5 minutes at 72 ° C. for 1 minute, 5 cycles PCR was performed to obtain a PCR product m3p in which m3F and m3R were ligated.

（ａ−４）（ａ−３）で得られたＰＣＲ産物ｍ３ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとしてＰＣＲを行なった。ＰＣＲは、表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行なった。これによりＦｃＲ２に１箇所アミノ酸置換を導入したＦｃＲ３をコードするポリヌクレオチドを作製した。

(A-4) The PCR product m3p obtained in (a-3) was used as a template, and PCR was performed using an oligonucleotide consisting of the sequence described in SEQ ID NO: 23 and SEQ ID NO: 24 as a PCR primer. After preparing the reaction solution having the composition shown in Table 7, the PCR is heat-treated at 98 ° C for 5 minutes, and the first step at 98 ° C for 10 seconds, the second step at 55 ° C for 5 seconds, 72 ° C. The reaction was performed for 30 cycles, with the third step of 1 minute being one cycle. Thus, a polynucleotide encoding FcR3 in which a single amino acid substitution was introduced into FcR2 was prepared.

（ａ−５）（ａ−４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１−２０６０４６号公報）にライゲーションし、これを用いて大腸菌Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）株を形質転換した。
（ａ−６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、野生型Ｆｃ結合性タンパク質に対して３箇所アミノ酸置換したポリペプチドである、ＦｃＲ３をコードするポリヌクレオチドを含むプラスミドｐＥＴ−ＦｃＲ３を得た。
（ａ−７）ｐＥＴ−ＦｃＲ３のヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。

(A-5) After purification of the polynucleotide obtained in (a-4), an expression vector pETMalE digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E. coli. E. coli BL21 (DE3) strain was transformed.
(A-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. By extracting a plasmid from the collected cells (transformants), a plasmid pET-FcR3 containing a polynucleotide encoding FcR3, which is a polypeptide having three amino acid substitutions for wild-type Fc binding protein, is obtained. The
(A-7) Analysis of the nucleotide sequence of pET-FcR3 was performed in the same manner as in Example 1 (5).

  The amino acid sequence of FcR3 to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 31 and the sequence of the polynucleotide encoding FcR3 is shown in SEQ ID NO: 32. In SEQ ID NO: 31, the 1st methionine (Met) to the 26th alanine (Ala) is a MalE signal peptide, the 27th lysine (Lys) to the 32nd methionine (Met) is a linker sequence, , Glycine (Gly) at position 33 to glutamine (Gln) at position 208 is the amino acid sequence of FcR3 (corresponding to the region from 17th to 192nd in SEQ ID NO: 1), glycine (Gly) from 209th to 210th Is a linker sequence, and histidine (His) at positions 211 to 216 is a tag sequence. In SEQ ID NO: 31, the glutamic acid of Val27Glu is at position 43, the asparagine of Tyr35Asn is at position 51, and the leucine of Phe75Leu is at position 91.

(B) FcR4
Among the amino acid substitutions involved in improving the stability of the Fc binding protein revealed in Example 3, Val27Glu, Tyr35Asn, Phe75Leu and Glu121Gly were selected, and these substitutions were integrated into the wild-type Fc binding protein FcR4 was produced. Specifically, FcR4 was produced by performing mutagenesis to generate Phe75Leu and Glu121Gly to a polynucleotide encoding FcR2.
The PCR product m3F was obtained by the method similar to (b-1) (a-1). Further, using as a template the plasmid expressing the Fc binding protein (Table 4) containing the amino acid substitutions of Ala71Asp, Phe75Leu and Glu121Gly obtained in Example 3 as oligonucleotides, oligonucleotides consisting of the sequences of SEQ ID NO: 24 and SEQ ID NO: 29 The PCR product m4R was obtained by performing PCR in the same manner as (a-1), using
(B-2) After mixing two types of PCR products (m3F and m4R) obtained by (b-1), PCR was performed by the same method as (a-3), and m3F and m4R were ligated. The resulting PCR product was designated m4p.
(B-3) A method similar to (a-4), using as a PCR primer an oligonucleotide consisting of the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 with the PCR product m4p obtained in (b-2) as a template PCR was performed. Thus, a polynucleotide encoding FcR4 was produced.
(B-4) After purification of the polynucleotide obtained in (b-3), an expression vector pETMalE digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E. coli. E. coli BL21 (DE3) strain was transformed.
(B-5) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. By extracting a plasmid from the collected cells (transformants), a plasmid pET-FcR4 containing a polynucleotide encoding FcR4, which is a polypeptide substituted at four amino acids with respect to a wild type Fc binding protein, is obtained. The
(B-6) Analysis of the nucleotide sequence of pET-FcR4 was carried out in the same manner as in Example 1 (5).

  The amino acid sequence of FcR4 to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 33, and the sequence of the polynucleotide encoding FcR4 is shown in SEQ ID NO: 34. In SEQ ID NO: 33, the first methionine (Met) to the 26th alanine (Ala) is a MalE signal peptide, and the 27th lysine (Lys) to the 32nd methionine (Met) is a linker sequence. From the 33rd glycine (Gly) to the 208th glutamine (Gln) corresponds to the amino acid sequence of FcR4 (corresponding to the 17th to 192th regions of SEQ ID NO: 1), the 209th to 210th glycines (Gly Is a linker sequence, and histidine (His) at positions 211 to 216 is a tag sequence. Further, in SEQ ID NO: 33, glutamic acid at Val27Glu is at position 43, asparagine at Tyr35Asn is at position 51, leucine at Phe75Leu is at position 911, and glycine at Glu121Gly is at position 137, respectively.

(C) FcR5a
Among the amino acid substitutions involved in improving the stability of the Fc binding protein revealed in Example 3, Val27Glu, Tyr35Asn, Phe75Leu, Asn92Ser and Glu121Gly are selected, and these substitutions are made into a wild type Fc binding protein. Accumulated FcR5a was generated. Specifically, FcR5a was produced by performing mutation to produce Asn92Ser to the polynucleotide encoding FcR4 produced in (b).
(C-1) (b), except that pET-FcR4 was used as a template, and the oligonucleotide consisting of the sequence described in SEQ ID NO: 22 and SEQ ID NO: 35 (5′-GAATATCGTTGCCAGACCAGCCAGCGACC-3 ′) was used as a PCR primer , PCR was performed in the same manner as (a-1). The purified PCR product was designated m5aF.
(C-2) With the pET-FcR4 prepared in (b) as a template, an oligonucleotide consisting of the sequence described in SEQ ID NO: 21 and SEQ ID NO: 36 (5'-GATCGCTCAGGGTGCTCAGGCTGGTCTGCGC-3 ') was used as a PCR primer except PCR was performed in the same manner as (a-1). The purified PCR product was called m5aR.
(C-3) After mixing the two types of PCR products (m5aF and m5aR) obtained in (c-1) and (c-2), PCR is performed in the same manner as in (a-3), m5aF And m5aR were linked. The resulting PCR product was designated m5ap.
(C-4) A method similar to (a-4), using as a PCR primer an oligonucleotide consisting of the sequences described in SEQ ID NO: 21 and SEQ ID NO: 22 with the PCR product m5ap obtained in (c-3) as a template PCR was performed. Thus, a polynucleotide encoding FcR5a was produced.
(C-5) After purification of the polynucleotide obtained in (c-4), an expression vector pETMalE digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E. coli. E. coli BL21 (DE3) strain was transformed.
(C-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. By extracting a plasmid from the collected cells (transformants), a plasmid pET-FcR5a containing a polynucleotide encoding FcR5a, which is a polypeptide substituted at five amino acids with respect to a wild type Fc binding protein, is obtained. The
(C-7) Analysis of the nucleotide sequence of pET-FcR5a was carried out in the same manner as in Example 1 (5).

  The amino acid sequence of FcR5a to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 37, and the sequence of the polynucleotide encoding FcR5a is shown in SEQ ID NO: 38. In SEQ ID NO: 37, the first methionine (Met) to the 26th alanine (Ala) is a MalE signal peptide, and the 27th lysine (Lys) to the 32nd methionine (Met) is a linker sequence. From the 33rd glycine (Gly) to the 208th glutamine (Gln) corresponds to the amino acid sequence of FcR 5a (corresponding to the 17th to 192th regions of SEQ ID NO: 1), the 209th to 210th glycines (Gly Is a linker sequence, and histidine (His) at positions 211 to 216 is a tag sequence. In SEQ ID NO: 37, Glutamic acid at Val27Glu is at position 43, asparagine at Tyr35Asn is at position 51, leucine at Phe75Leu is at position 91, serine at Asn92Ser is at position 108, and glycine for Glu121Gly is at position 137.

(D) FcR5b
Among the amino acid substitutions involved in improving the stability of the Fc binding protein revealed in Example 3, Val27Glu, Tyr35Asn, Glu54Asp, Phe75Leu and Glu121Gly are selected, and these substitutions are made into a wild type Fc binding protein. Accumulated FcR5b was generated. Specifically, FcR5b was prepared by performing mutagenesis to generate Glu54Asp to the polynucleotide encoding FcR4 prepared in (b).
(D-1) (b), except that pET-FcR4 was used as a template, and an oligonucleotide consisting of the sequence described in SEQ ID NO: 22 and SEQ ID NO: 39 (5′-CAGGGCGCGTATAGCCGGATGATAACAGC-3 ′) was used as a PCR primer , PCR was performed in the same manner as (a-1). The purified PCR product was called m5bF.
(D-2) With the pET-FcR4 prepared in (b) as a template, an oligonucleotide consisting of the sequence described in SEQ ID NO: 21 and SEQ ID NO: 40 (5′-CACTGGGTGCTGTTATCATCCGGGCTATAC-3 ′) was used as a PCR primer except PCR was performed in the same manner as (a-1). The purified PCR product was called m5bR.
(D-3) After mixing the two PCR products (m5bF and m5bR) obtained in (d-1) and (d-2), PCR is carried out in the same manner as in (a-3) to obtain m5bF and m5bF The m5bR was ligated. The obtained PCR product was called m5 bp.
(D-4) A method similar to (a-4), using as a PCR primer an oligonucleotide consisting of the sequences described in SEQ ID NO: 21 and SEQ ID NO: 22 with the PCR product m5 bp obtained in (d-3) as a template PCR was performed. Thus, a polynucleotide encoding FcR5b was produced.
(D-5) After purification of the polynucleotide obtained in (d-4), an expression vector pETMalE digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E. coli. E. coli BL21 (DE3) strain was transformed.
(D-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. By extracting a plasmid from the collected cells (transformants), a plasmid pET-FcR5b containing a polynucleotide encoding FcR5b, which is a polypeptide substituted at five amino acids with respect to the wild type Fc binding protein, is obtained. The
(D-7) Analysis of the nucleotide sequence of pET-FcR5b was carried out in the same manner as in Example 1 (5).

  The amino acid sequence of FcR5b to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 41, and the sequence of the polynucleotide encoding FcR5b is shown in SEQ ID NO: 42. In SEQ ID NO: 41, the 1st methionine (Met) to the 26th alanine (Ala) is a MalE signal peptide, and the 27th lysine (Lys) to the 32nd methionine (Met) is a linker sequence. From the 33rd glycine (Gly) to the 208th glutamine (Gln) corresponds to the amino acid sequence of FcR 5b (corresponding to the 17th to 192th regions of SEQ ID NO: 1), the 209th to 210th glycines (Gly Is a linker sequence, and histidine (His) at positions 211 to 216 is a tag sequence. Further, in SEQ ID NO: 41, Glutamic acid at Val27Glu is at position 43, Asparagine at Tyr35Asn is at position 51, aspartic acid for Glu54Asp is at position 70, leucine at Phe75Leu is at position 91, and glycine at Glu121Gly is at position 137, respectively.

(E) FcR6a
Among the amino acid substitutions involved in improving the stability of the Fc binding protein revealed in Example 3, Val27Glu, Tyr35Asn, Glu54Asp, Phe75Leu, Asn92Ser and Glu121Gly are selected, and these substitutions are wild-type Fc binding. We prepared FcR6a accumulated in proteins. Specifically, FcR6a was produced by performing mutagenesis to generate Glu54Asp to the polynucleotide encoding FcR5a produced in (c).
(E-1) The same method as (a-1), except that the pET-FcR5a prepared in (c) was used as a template and oligonucleotides consisting of the sequences of SEQ ID NO: 22 and SEQ ID NO: 39 were used as PCR primers. PCR was performed. The purified PCR product was called m6aF.
(E-2) The same method as (a-1), except that the pET-FcR4 prepared in (b) was used as a template and oligonucleotides consisting of the sequences of SEQ ID NO: 21 and SEQ ID NO: 40 were used as PCR primers. PCR was performed. The purified PCR product was called m6aR.
(E-3) After mixing the two types of PCR products (m6aF and m6aR) obtained in (e-1) and (e-2), PCR is performed in the same manner as in (a-3) to obtain m6aF and m6aF m6aR was linked. The obtained PCR product was called m6ap.
(E-4) A method similar to (a-4), using as a PCR primer an oligonucleotide consisting of the sequence described in SEQ ID NO: 21 and SEQ ID NO: 22 with the PCR product m6ap obtained in (e-3) as a template PCR was performed. Thus, a polynucleotide encoding FcR6a was produced.
(E-5) After purification of the polynucleotide obtained in (e-4), an expression vector pETMalE digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E. coli. E. coli BL21 (DE3) strain was transformed.
(E-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. By extracting a plasmid from the recovered cells (transformants), a plasmid pET-FcR6a containing a polynucleotide encoding FcR6a, which is a polypeptide substituted at six amino acids with respect to a wild type Fc binding protein, is obtained. The
(E-7) Analysis of the nucleotide sequence of pET-FcR6a was carried out in the same manner as in Example 1 (5).

  The amino acid sequence of FcR6a to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 43, and the sequence of the polynucleotide encoding FcR6a is shown in SEQ ID NO: 44. In SEQ ID NO: 43, the first methionine (Met) to the 26th alanine (Ala) is a MalE signal peptide, and the 27th lysine (Lys) to the 32nd methionine (Met) is a linker sequence. From the 33rd glycine (Gly) to the 208th glutamine (Gln) corresponds to the amino acid sequence of FcR 6a (corresponding to the 17th to 192th regions of SEQ ID NO: 1), the 209th to 210th glycines (Gly Is a linker sequence, and histidine (His) at positions 211 to 216 is a tag sequence. In SEQ ID NO: 43, the glutamic acid of Val27Glu is at position 43, the asparagine of Tyr35Asn is at position 51, the aspartic acid of Glu54Asp is at position 70, the leucine of Phe75Leu is at position 91, the serine at Asn92Ser is at position 108, and the glycine of Glu121Gly is at position 137 Each exists in

(F) FcR6b
Among the amino acid substitutions involved in the improvement of the stability of the Fc binding protein revealed in Example 3, Val27Glu, Tyr35Asn, Glu54Asp, Phe75Leu, Glu120Val and Glu121Gly are selected, and these substitutions are wild-type Fc binding Protein-accumulated FcR6b was produced. Specifically, FcR6b was produced by performing mutagenesis to generate Glu120Val to the polynucleotide encoding FcR5b produced in (d).
With the pET-FcR5b prepared in (f-1) (d) as a template, an oligonucleotide consisting of the sequence described in SEQ ID NO: 22 and SEQ ID NO: 45 (5′-GTGTTCAAAGTGGGGGATCCGGATTCATCTG-3 ′) was used as a PCR primer except PCR was performed in the same manner as (a-1). The purified PCR product was called m6bF.
(F-2) (p) using the pET-FcR5b prepared in (d) as a template and the oligonucleotide consisting of the sequence described in SEQ ID NO: 21 and SEQ ID NO: 46 (5'-AATCGGATCCCCCCACTTTGAACACCCACCG-3 ') as PCR primers, PCR was performed in the same manner as (a-1). The purified PCR product was called m6bR.
(F-3) After mixing the two types of PCR products (m6bF and m6bR) obtained in (f-1) and (f-2), PCR is performed in the same manner as in (a-3) to obtain m6bF and m6bF The m6bR was ligated. The obtained PCR product was called m6 bp.
(F-4) The PCR product m6 bp obtained in (f-3) was used as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 21 and SEQ ID NO: 22 were used as PCR primers, except for (a-4) PCR was performed in the same manner. Thus, a polynucleotide encoding FcR6b was produced.
(F-5) After purification of the polynucleotide obtained in (f-4), an expression vector pETMalE digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E. coli. E. coli BL21 (DE3) strain was transformed.
(F-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. By extracting a plasmid from the collected cells (transformants), a plasmid pET-FcR6b containing a polynucleotide encoding FcR6b, which is a polypeptide substituted at six amino acids with respect to a wild type Fc binding protein, is obtained. The
(F-7) Analysis of the nucleotide sequence of pET-FcR6b was performed in the same manner as in Example 1 (5).

  The amino acid sequence of FcR6b to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 47, and the sequence of the polynucleotide encoding FcR6b is shown in SEQ ID NO: 48. In SEQ ID NO: 47, the first methionine (Met) to the 26th alanine (Ala) is a MalE signal peptide, and the 27th lysine (Lys) to the 32nd methionine (Met) is a linker sequence. From the 33rd glycine (Gly) to the 208th glutamine (Gln) corresponds to the amino acid sequence of FcR6b (corresponding to the 17th to 192th regions of SEQ ID NO: 1), the 209th to 210th glycines (Gly Is a linker sequence, and histidine (His) at positions 211 to 216 is a tag sequence. Further, in SEQ ID NO: 47, the glutamate at Val27Glu is at position 43, the asparagine at Tyr35Asn is at position 51, the aspartic acid at Glu54Asp is at position 70, the leucine at Phe75Leu is at position 91, the valine at Glu120Val is at position 136, and the glycine at Glu121Gly is at position 137 Each exists in

(G) FcR7
Among the amino acid substitutions involved in the improvement of the stability of the Fc-binding protein revealed in Example 3, Val27Glu, Tyr35Asn, Glu54Asp, Phe75Leu, Asn92Ser, Glu120Val and Glu121Gly are selected, and those substitutions are wild-type Fc FcR7 accumulated in binding protein was prepared. Specifically, FcR7 was produced by performing mutagenesis to generate Glu120Val to the polynucleotide encoding FcR6a produced in (e).
(G-1) A method similar to (a-1), except that the pET-FcR6a prepared in (e) was used as a template and oligonucleotides consisting of the sequences described in SEQ ID NO: 22 and SEQ ID NO: 45 were used as PCR primers. PCR was performed. The purified PCR product was designated m7F.
(G-2) A method similar to (a-1), except that the pET-FcR6a prepared in (e) was used as a template and oligonucleotides consisting of the sequences described in SEQ ID NO: 21 and SEQ ID NO: 46 were used as PCR primers. PCR was performed. The purified PCR product was designated m7R.
(G-3) After mixing the two PCR products (m7F and m7R) obtained in (g-1) and (g-2), PCR is carried out in the same manner as in (a-3), m7F And m7R were linked. The resulting PCR product was designated m7p.
(G-4) The PCR product m7p obtained in (g-3) was used as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 21 and SEQ ID NO: 22 were used as PCR primers, except for (a-4) A similar PCR was performed. Thus, a polynucleotide encoding FcR7 was produced.
(G-5) After purification of the polynucleotide obtained in (g-4), an expression vector pETMalE digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E. coli. E. coli BL21 (DE3) strain was transformed.
(G-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. By extracting a plasmid from the collected cells (transformants), a plasmid pET-FcR7 containing a polynucleotide encoding FcR7, which is a polypeptide having 7 amino acid substitutions for wild-type Fc binding protein, is obtained. The
(G-7) Analysis of the nucleotide sequence of pET-FcR7 was performed in the same manner as in Example 1 (5).

The amino acid sequence of FcR7 to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 49, and the sequence of the polynucleotide encoding FcR7 is shown in SEQ ID NO: 50. In SEQ ID NO: 49, the first methionine (Met) to the 26th alanine (Ala) is a MalE signal peptide, and the 27th lysine (Lys) to the 32nd methionine (Met) is a linker sequence. The 33rd glycine (Gly) to the 208th glutamine (Gln) are the amino acid sequence of FcR7 (corresponding to the 17th to 192th regions of SEQ ID NO: 1), the 209th to 210th glycines (Gly) Is a linker sequence, and histidine (His) at positions 211 to 216 is a tag sequence. Further, in SEQ ID NO: 49, glutamic acid of Val27Glu is 43rd, asparagine of Tyr35Asn is 51st, aspartic acid of Glu54Asp is 70th, leucine of Phe75Leu is 91st, serine of Asn92Ser is 108th, valine of Glu120Val is 136th, Glu121Gly Glycines are present at position 137, respectively.

Example 5 Thermal stability evaluation of modified Fc binding protein (1) Wild type Fc binding protein prepared in Example 1, Fc binding protein of mutant selected in Example 3 (FcR2), and Example 4 The transformant expressing the mutant Fc binding protein (FcR3, FcR4, FcR5a, FcR5b, FcR6a, FcR6b, FcR7) prepared in above was inoculated into 3 mL of 2YT liquid medium containing 50 μg / mL of kanamycin, respectively. Preculture was performed by aerobic shaking culture overnight at 37 ° C.
(2) Inoculate 200 μL of the preculture liquid into 20 mL of 2YT liquid medium (Peptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L) supplemented with 50 μg / mL kanamycin, and aerobically at 37 ° C Shake culture was performed.
(3) After 1.5 hours from the start of culture, the culture temperature was changed to 20 ° C., and shake culture was performed for 30 minutes. Thereafter, IPTG was added to a final concentration of 0.01 mM, and aerobic culture was continued at 20 ° C. overnight.
(4) After completion of the culture, cells were collected by centrifugation, and a protein extract was prepared using BugBuster Protein extraction kit (manufactured by Takara Bio Inc.).
(5) The antibody binding activity of the wild type Fc binding protein and the mutant Fc binding protein in the protein extract prepared in (4) was measured using the ELISA method described in Example 3 (4) . At this time, a calibration curve was prepared using a commercially available extracellular region of FcγRIIIa (R & D Systems: 4325-FC-050), and concentration measurement was performed.
(6) Dilution was performed with 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride so that the concentration of each protein was 5 μg / mL. This was divided into equal amounts, and one was heat-treated at 45 ° C. for 10 minutes using a thermal cycler (manufactured by Eppendorf), and the other was not heat-treated. The antibody binding activity of the heat-treated or non-heat-treated protein is measured by the ELISA method described in Example 3 (4), and the antibody binding activity when heat-treated is the antibody binding activity when not heat-treated. The residual activity was calculated by dividing by.

  The results are shown in Table 8. The mutant Fc binding protein (FcR2, FcR3, FcR4, FcR5a, FcR5b, FcR6a, FcR6b, FcR7) evaluated here has higher residual activity compared to the wild type Fc binding protein, and the mutant It was confirmed that the thermal stability of the Fc binding protein of

実施例６ 改良Ｆｃ結合性タンパク質の酸安定性評価
（１）実施例５（１）から（５）と同様の方法で改良Ｆｃ結合性タンパク質を調製した。
（２）各タンパク質の濃度が３０μｇ／ｍＬになるよう１５０ｍＭの塩化ナトリウムを含んだ２０ｍＭのトリス緩衝液（ｐＨ７．４）で希釈した。希釈した各Ｆｃ結合性タンパク質６０μＬと０．１Ｍのグリシン塩酸緩衝液（ｐＨ３．０）１２０μＬを混合し、３０℃で２時間静置した。
（３）実施例３（４）に記載のＥＬＩＳＡ法によって、グリシン塩酸緩衝液（ｐＨ３．０）による酸処理を行なった後のタンパク質の抗体結合活性と、前記酸処理を行なわなかったときのタンパク質の抗体結合活性を測定した。その後、酸処理を行なった場合の抗体結合活性を、酸処理を行なわなかったときの抗体結合活性で除することで、残存活性を算出した。

Example 6 Evaluation of Acid Stability of Modified Fc-Binding Protein (1) A modified Fc-binding protein was prepared in the same manner as in Example 5 (1) to (5).
(2) Dilution was carried out with 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride so that the concentration of each protein would be 30 μg / mL. 60 μL of each diluted Fc binding protein and 120 μL of 0.1 M glycine hydrochloride buffer (pH 3.0) were mixed and allowed to stand at 30 ° C. for 2 hours.
(3) Antibody binding activity of the protein after acid treatment with glycine hydrochloride buffer (pH 3.0) by the ELISA method described in Example 3 (4), and protein when the acid treatment was not performed Antibody binding activity was measured. Thereafter, the residual activity was calculated by dividing the antibody binding activity when acid treatment was performed by the antibody binding activity when acid treatment was not performed.

  The results are shown in Table 9. The mutant Fc binding protein (FcR2, FcR3, FcR4, FcR5a, FcR5b, FcR6a, FcR6b, FcR7) evaluated here has higher residual activity compared to the wild type Fc binding protein, and the mutant It was confirmed that the acid stability of the Fc binding protein of

実施例７ １箇所アミノ酸置換したＦｃ結合性タンパク質の作製
実施例３で明らかになったＦｃ結合性タンパク質の安定性向上に関与するアミノ酸置換のうち、配列番号１の２７番目のバリン（Ｖａｌ）、３５番目のチロシン（Ｔｙｒ）および１２１番目のグルタミン酸（Ｇｌｕ）について、他のアミノ酸に置換したＦｃ結合性タンパク質を、それぞれ下記の方法で作製した。

Example 7 Preparation of Fc binding protein substituted with single amino acid substitution Among the amino acid substitutions involved in the improvement of stability of Fc binding protein revealed in Example 3, valine at position 27 of SEQ ID NO: 1 (Val), For the 35th tyrosine (Tyr) and the 121st glutamic acid (Glu), Fc-binding proteins substituted with other amino acids were produced by the following methods, respectively.

(A) Preparation of an Fc binding protein in which valine (Val) at position 27 of SEQ ID NO: 1 is substituted with another amino acid (A-1) SEQ ID NO: 24 and sequence using the pET-eFcR prepared in Example 1 as a template PCR was performed in the same manner as in Example 4 (a-1), except that the oligonucleotide consisting of the sequence described in No. 51 (5'-CTGCCCAAAAGCGNNKGTGTTTCTGGAACCG-3 ') was used as a PCR primer. The purified PCR product was 27 pF.
(A-2) With the pET-eFcR prepared in Example 1 as a template, an oligonucleotide consisting of the sequence described in SEQ ID NO: 23 and SEQ ID NO: 52 (5′-TTCCAGAAACACMNNCGCTTTCGGCAGATC-3 ′) was used as a PCR primer except PCR was performed in the same manner as in Example 4 (a-1). The purified PCR product was 27 pR.
(A-3) After mixing the two PCR products (27 pF, 27 pR) obtained in (A-1) and (A-2), PCR was carried out in the same manner as in Example 4 (a-3). , 27 pF and 27 pR were linked. The obtained PCR product was called 27p.
(A-4) With the PCR product 27p obtained in (A-3) as a template, oligonucleotides consisting of the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers and Example 4 (a-4) PCR was performed in the same manner. Thus, a polynucleotide encoding an Fc binding protein was prepared in which valine at position 27 of SEQ ID NO: 1 was substituted with any amino acid.
(A-5) After purification of the polynucleotide obtained in (A-4), the expression vector pETMalE digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E. coli. E. coli BL21 (DE3) strain was transformed.
(A-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. A plasmid was extracted from the collected cells (transformants), and nucleotide sequence analysis was performed in the same manner as in Example 1 (5).

  As a result, a polynucleotide encoding an Fc binding protein in which amino acid substitution of Val27Gly (V27G), Val27Lys (V27K), Val27Thr (V27T), Val27Ala (V27A), Val27Trp (V27W), or Val27Arg (V27R) occurred was obtained. .

(B) Preparation of an Fc binding protein in which tyrosine (Tyr) at position 35 of SEQ ID NO: 1 is substituted with another amino acid (B-1) SEQ ID NO: 24 and sequence using the pET-eFcR prepared in Example 1 as a template PCR was performed in the same manner as in Example 4 (a-1), except that the oligonucleotide consisting of the sequence described in No. 53 (5'-AACCGCAGTGGNNKCGCGTGCTGGAGAAAG-3 ') was used as a PCR primer. The purified PCR product was 35 pF.
(B-2) With the pET-eFcR prepared in Example 1 as a template, an oligonucleotide consisting of the sequence described in SEQ ID NO: 23 and SEQ ID NO: 54 (5′-AGCACAGCGMNNCCACTGCGGTTCCAGAAAC-3 ′) was used as a PCR primer except PCR was performed in the same manner as in Example 4 (a-1). The purified PCR product was 35 pR.
(B-3) After mixing the two PCR products (35 pF, 35 pR) obtained in (B-1) and (B-2), PCR was carried out in the same manner as in Example 4 (a-3). Conducted and connected 35 pF and 35 pR. The resulting PCR product was 35 p.
(B-4) Using the PCR product 35p obtained in (B-3) as a template, and an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 23 and SEQ ID NO: 24 as a PCR primer, Example 4 (a-4) PCR was performed in the same manner. Thus, a polynucleotide encoding an Fc binding protein was prepared in which the 35th tyrosine of SEQ ID NO: 1 was substituted with any amino acid.
(B-5) After purification of the polynucleotide obtained in (B-4), the expression vector pETMalE digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E. coli. E. coli BL21 (DE3) strain was transformed.
(B-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. A plasmid was extracted from the collected cells (transformants), and nucleotide sequence analysis was performed in the same manner as in Example 1 (5).

  As a result, Tyr35Cys (Y35C), Tyr35Asp (Y35D), Tyr35Phe (Y35F), Tyr35Gly (Y35G), Tyr35Lys (Y35K), Tyr35Leu (Y35L), Tyr35Asu (Y35L), Tyr35Pro (Y35P), Tyr35Arg (Y35R), ), A polynucleotide encoding an Fc binding protein in which amino acid substitution of Tyr35Thr (Y35T), Tyr35Val (Y35V), or Tyr35Trp (Y35W) has occurred.

(C) F in which the 121st glutamic acid (Glu) of SEQ ID NO: 1 is substituted with another amino acid
c Preparation of c-Binding Protein (C-1) Using pET-eFcR prepared in Example 1 as a template, an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 24 and SEQ ID NO: 55 (5′-GTGTTCAAAGAGNNKGATCC GATTCATCTG-3 ′) is PCR PCR was performed in the same manner as in Example 4 (a-1) except for using as a primer. The purified PCR product was 121 pF.
(C-2) With the pET-eFcR prepared in Example 1 as a template, an oligonucleotide consisting of the sequence described in SEQ ID NO: 23 and SEQ ID NO: 56 (5'-AATCGGATCMNNCTCTTTGAACACCCACCG-3 ') was used as a PCR primer except PCR was performed in the same manner as in (a-1) of Example 4. The purified PCR product was called 121 pR.
(C-3) After mixing the two PCR products (121 pF, 121 pR) obtained by (C-1) and (C-2), PCR was carried out in the same manner as in Example 4 (a-3). Conducted and linked 121 pF and 121 pR. The resulting PCR product was designated 121p.
(C-4) The PCR product 121p obtained in (C-3) is used as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 are used as PCR primers in the same manner as in Example 4 (a-4) PCR was performed. Thus, a polynucleotide encoding an Fc binding protein in which glutamic acid at position 121 of SEQ ID NO: 1 was substituted with any amino acid was produced.
(C-5) After purification of the polynucleotide obtained in (C-4), it is digested with restriction enzymes NcoI and HindIII, and digested with restriction enzymes NcoI and HindIII into expression vector pETMalE (Japanese Patent Laid-Open No. 2011-206046). After ligation, E. coli E. coli was used. E. coli BL21 (DE3) strain was transformed.
(C-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. A plasmid was extracted from the collected cells (transformants), and nucleotide sequence analysis was performed in the same manner as in Example 1 (5).

  As a result, a polynucleotide encoding an Fc binding protein in which amino acid substitution of Glu121Lys (E121K), Glu121Pro (E121P), Glu121Arg (E121R), Glu121Gly (E121G), Glu121His (E121H), or Glu121Val (E121V) occurred was obtained. .

Example 8 Evaluation of Antibody Binding Activity of One Amino Acid-Substituted Fc-Binding Protein (1) The wild-type Fc-binding protein prepared in Example 1 and the Fc-binding protein with one amino acid substitution prepared in Example 7 are expressed The transformant was cultured in the same manner as in Example 3 (1) and (2) to express a wild-type Fc binding protein and an Fc binding protein with 1 amino acid substitution.
(2) The binding activity to the antibody was examined by the ELISA method described in Example 3 (4), for the expressed 1 amino acid substituted Fc binding protein.

  The results are shown in FIG. An antibody compared with a wild type Fc binding protein by substituting valine at position 27 of SEQ ID NO: 1 with glycine (V27G), lysine (V27K), threonine (V27T), alanine (V27A), arginine (V27R) While the binding activity was improved, substitution of Val at position 27 of SEQ ID NO: 1 with tryptophan (V27W) reduced antibody binding activity as compared to wild-type Fc binding protein.

  The 35th tyrosine of SEQ ID NO: 1 is aspartic acid (Y35D), phenylalanine (Y35F), glycine (Y35G), lysine (Y35K), leucine (Y35L), asparagine (Y35N), proline (Y35P), serine (Y35S) By substituting threonine (Y35T), valine (Y35V) and tryptophan (Y35W), the antibody binding activity was improved as compared to the wild type Fc binding protein. Among them, Y35D, Y35G, Y35K, Y35L, Y35N, Y35P, Y35S, Y35T, and Y35W significantly improved the antibody binding activity as compared to the wild type Fc binding protein. On the other hand, when the 35th tyrosine of SEQ ID NO: 1 was substituted with cysteine (Y35C) or arginine (Y35R), the antibody binding activity was almost equivalent to that of a wild type Fc binding protein.

  By substituting glutamic acid at position 121 of SEQ ID NO: 1 with lysine (E121 K), arginine (E121 R), glycine (E121 G), and histidine (E121 H), antibody binding activity was improved compared to wild-type Fc binding protein . Among them, E121G has significantly improved antibody binding activity as compared to wild type Fc binding protein. On the other hand, glutamic acid at position 121 of SEQ ID NO: 1 is substituted with valine (E121V) for antibody binding activity almost equivalent to that of wild type Fc binding protein, and when substituted for proline (E121 P), wild type Fc binding The antibody binding activity was reduced compared to the sex protein.

Example 9 Thermal stability evaluation of 1 amino acid substituted Fc binding protein In order to compare the thermal stability of 1 amino acid substituted Fc binding protein evaluated in Example 8, heat treatment is carried out in the same manner as in Example 3 (3) Was carried out (45.degree. C., 10 minutes) to calculate the residual activity.

  The results are shown in FIG. The 35th tyrosine of SEQ ID NO: 1 is aspartic acid (Y35D), glycine (Y35G), lysine (Y35K), leucine (Y35L), asparagine (Y35N), proline (Y35P), serine (Y35S), threonine (Y35T) The Fc binding protein substituted for each and the Fc binding protein obtained by substituting glutamic acid at position 121 of SEQ ID NO: 1 with glycine (E121G) have significantly improved thermal stability compared to the wild type Fc binding protein . Among them, Y35N and Y35P significantly improved the thermal stability as compared to the wild type Fc binding protein.

Example 10 Large-scale preparation of FcR5a (1) 400 mL of 2YT liquid medium containing 50 μg / mL kanamycin (Peptone 16 g / mL in a 2 L baffle flask, the transformant expressing FcR5 a prepared in Example 4 (c) L., yeast extract 10 g / L, sodium chloride 5 g / L), and preculture was performed by aerobic shaking culture overnight at 37 ° C.
(2) Glucose 10 g / L, yeast extract 20 g / L, trisodium phosphate dodecahydrate 3 g / L, disodium hydrogen phosphate dodecahydrate 9 g / L, ammonium chloride 1 g / L and kanamycin sulfate 50 mg 180 mL of the culture solution of (1) was inoculated into 1.8 L of liquid medium containing L / L, and main culture was performed using a 3 L fermenter (manufactured by Biot). The main culture was started under conditions of a temperature of 30 ° C., a pH of 6.9 to 7.1, an aeration rate of 1 VVM, and a dissolved oxygen concentration of 30% saturation concentration. For pH control, 50% phosphoric acid as acid and 14% ammonia water as alkali were used respectively. Control of dissolved oxygen was controlled by changing the stirring speed, and the rotation speed was set to the lower limit of 500 rpm and the upper limit of 1000 rpm . After the start of culture, when glucose concentration can not be measured, feed medium (248.9 g / L glucose, 83.3 g / L yeast extract, 7.2 g / L magnesium sulfate heptahydrate) dissolved oxygen (DO Under control).
(3) As a measure of the amount of cells, when the absorbance at 600 nm (OD _{600 nm} ) reaches about 150, the culture temperature is lowered to 25 ° C. and after confirming that the set temperature has been reached, the final concentration becomes 0.5 mM Then, IPTG was added, and culture was continued at 25 ° C.
(4) Stop the culture about 48 hours after the start of the culture, and culture the medium at 8000 rpm at 4 ° C, 20
The cells were collected by centrifugation for a minute.
(5) A portion of the cells recovered in (4) is suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride so as to be 5 mL / 1 g (cells) to generate ultrasonic waves. The cells were disrupted at an output of about 150 W for about 10 minutes at 4 ° C. using an apparatus (Insonator 201M (trade name), manufactured by Kubota Corporation). The disrupted cell suspension was centrifuged twice at 10,000 rpm for 20 minutes at 4 ° C., and the supernatant was recovered.
(6) After adding imidazole to a final concentration of 20 mM to the disrupted solution obtained in (5), equilibrate with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole in advance The solution was applied to an XK 26/20 column (manufactured by GE Healthcare) packed with 50 mL of the obtained Ni Sepharose 6 Fast Flow (manufactured by GE Healthcare). After washing with the buffer used for equilibration, FcR5a was eluted using 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 0.5 M imidazole.
(7) HR loaded with 30 mL of IgG sepharose (manufactured by GE Healthcare) equilibrated with 20 mM Tris-HCl buffer solution (pH 7.4) containing 150 mM sodium chloride in advance. Applied to a 16/10 column (manufactured by GE Healthcare). After washing with the buffer used for equilibration, FcR5a was eluted with 0.1 M glycine hydrochloride buffer (pH 3.0). The pH of the eluate was returned to near neutrality by adding 1⁄4 volume of 1 M Tris-HCl buffer (pH 7.0).

Example 11 Preparation of FcR5a-immobilized gel (1) The FcR5a prepared in Example 10 is concentrated and buffer-exchanged using an ultrafiltration membrane (manufactured by Millipore: Amicon Ultra-15) to obtain 150 mM sodium chloride Concentrated in 20 mM Tris-HCl buffer (pH 7.4) to a concentration of 8.37 mg / mL.
(2) An epoxy Toyopearl gel was prepared by reacting 1,6-hexanediol diglycidyl ether with the hydroxyl group of a hydrophilic vinyl polymer (Tosoh Corporation: Toyopearl) as a carrier.
(3) Five spin columns (manufactured by Bio-Rad) containing 100 μL of the epoxy Toyopearl gel prepared in (2) were prepared, and 0.1 M borate buffer (pH 9) containing 0.5 M sodium chloride .0) Washed 3 times with 0.5 mL.
(4) A mixed solution of 0.3 mL of the FcR5a solution prepared in (1) and 0.45 mL of 0.1 M borate buffer (pH 9.0) containing 0.5 M sodium chloride, (3) Each was added to a spin column packed with the gel described in and shaken at 35 ° C. for 3 hours.
(5) After recovering a mixed solution of FcR5a solution added to the gel and 0.1 M borate buffer containing 0.5 M sodium chloride, 0.1 M glycine hydrochloride buffer (pH 3.0) 0. Wash 3 times with 2 mL. Thereafter, the pH was returned to near neutrality by washing three times with 0.5 mL of 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride to prepare 0.5 mL of FcR5a-immobilized gel.

  The concentration of protein contained in the solution recovered in (5) and the washing solution was measured, and the amount of FcR5a immobilized on the gel was calculated to calculate the percentage of immobilization: 33.7% of the added FcR5a Were immobilized on the gel.

Example 12 Antibody separation on FcR5a-immobilized gel (1) 0.5 mL of the FcR5a-immobilized gel prepared in Example 11 is packed in an HR16 / 5 column (manufactured by GE Healthcare), AKTAprime plus (GE Healthcare) Made). Thereafter, it was equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride.
(2) Flow 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride at a flow rate of 0.1 mL, and 1 mg with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride 0.5 mL of human IgG1 (Sigma: I5154-1MG) solution adjusted to / mL was applied, and human IgG1 was adsorbed to FcR5a immobilized gel. Thereafter, the plate was equilibrated by flowing 20 mM acetate buffer (pH 5.0), and the adsorbed human IgG1 was eluted with a pH gradient with 20 mM glycine hydrochloric acid buffer (pH 3.0). Fractions of eluted human IgG1 were collected every 0.5 mL.

  The elution pattern of human IgG1 and the collected fractions are shown in FIG. Among the eluted fractions collected, a mixture of fraction 13 (Fr13) and fraction 14 (Fr14) is designated fraction A (FrA), and a mixture of fraction 16 (Fr16) and fraction 17 (Fr17) is fraction B (FrB). ).

Example 13 Sugar chain structural analysis of isolated antibody (1) Human IgG1 and eluted fractions (FrA, FrB) before purification used in Example 12 are denatured by heat treatment at 100 ° C. for 10 minutes to obtain glycoamidase A / pepsin and They were sequentially treated with pronase and purified by gel filtration to obtain a sugar chain fraction.
(2) The sugar chain obtained in (1) is concentrated and dried with an evaporator, then 2-aminopyridine and then dimethylamine borane are sequentially reacted in an acetic acid solvent to form a fluorescently labeled sugar chain, and gel filtration is performed. Refined.
(3) Using the anion exchange column (TSKgel DEAE-5PW, φ 7.5 mm × 7.5 cm: Tosoh Corporation), the neutral sugar chain fraction and monosialylation of the fluorescently labeled sugar chain obtained in (2) It separated into a sugar chain fraction.
(4) The neutral sugar chain fraction and the monosialylated sugar chain fraction obtained in (3) were isolated into individual sugar chains using an ODS column. After acquiring molecular weight information of the sugar chain isolated by MALDI-TOF-MS analysis, the sugar chain structure was assigned in light of the retention time of the ODS column chromatograph.

  The compositional ratio of the neutral sugar chain is shown in Table 10, and the compositional ratio of the monosialylated sugar chain is shown in Table 11. In addition, assigned sugar chain structures (N1 to N8 and M1 and M2) are shown in FIG. From the results shown in Table 10, antibodies having sugar chain structures N2 and N7 were detected before purification and with FrB, but were not detected from FrA. That is, antibodies having the above two types of sugar chain structures (N2 and N7 in FIG. 5) were not detected in the early eluting fraction FrA, but were detected in the late eluting fraction FrB. It was shown to bind strongly to the FcR5a-immobilized gel as compared to an antibody having a sugar chain structure of From the above results, it was found that the FcR5a-immobilized gel, which is one aspect of the adsorbent of the present invention, can separate the antibody due to the difference in the sugar chain structure of the antibody.

実施例１４ ＦｃＲ５ａ固定化ゲルでの抗体精製
実施例１１で作製したＦｃＲ５ａ固定化ゲルを用いて、ヒトＩｇＧ１およびヒトＩｇＧ３の精製を行なった。
（１）実施例１１と同様の方法でＦｃＲ５ａ固定化ゲル０．２ｍＬを作製した。作製したＦｃＲ５ａ固定化ゲル０．１ｍＬをスピンカラムに充填した。
（２）（１）で作製したＦｃＲ５ａ固定化ゲルを充填したカラムを１５０ｍＭの塩化ナトリウムを含んだ２０ｍＭのトリス塩酸緩衝液（ｐＨ７．４）０．５ｍＬで３回洗浄した。
（３）動物細胞用の培地であるＤＭＥＭ／Ｆ１２培地（ライフテクノロジー社製）に１０％のウシ胎児血清（ＦＣＳ）を添加した培地に対し、ヒトＩｇＧ１（シグマ社製：Ｉ５１５４−１ＭＧ）またはヒトＩｇＧ３（シグマ社製：Ｉ４６３９−１ＭＧ）を０．３ｍｇ／ｍＬとなるように添加することで模擬培養液を調製した。
（４）（２）で洗浄したカラムに（３）で調製した模擬培養液を０．４ｍＬ加え、２５℃で２時間振とうした。
（５）模擬培養液を取り除き、１５０ｍＭの塩化ナトリウムを含んだ２０ｍＭのトリス塩酸緩衝液（ｐＨ７．４）０．５ｍＬで４回洗浄後、５００μＬの０．１Ｍのグリシン塩酸緩衝液（ｐＨ３．０）で溶出し、１００μＬごとにフラクションを回収した。
（６）模擬培養液、溶出フラクションをそれぞれ２×サンプルバッファー（２（ｗ／ｖ）％ドデシル硫酸ナトリウム、６（ｗ／ｖ）％β−メルカプトエタノール、１０（ｗ／ｖ）％グリセリンおよび０．００５（ｗ／ｖ）％ブロモフェノールブルーを含む５０ｍＭのトリス塩酸緩衝液（ｐＨ６．８））を添加し加熱処理した。その後、処理したサンプルを５から２０％のグラジエントＳＤＳ−ＰＡＧＥ用ゲル（アトー社製）を用いた電気泳動にて分離した。なお比較として、模擬培養液に添加したヒトＩｇＧ１およびヒトＩｇＧ３（濃度：０．２ｍｇ／ｍＬ）についても同様の処理を行ない、ＳＤＳ−ＰＡＧＥで分析した。

Example 14 Antibody Purification on FcR5a-Immobilized Gel The FcR5a-immobilized gel prepared in Example 11 was used to purify human IgG1 and human IgG3.
(1) In the same manner as in Example 11, 0.2 mL of FcR5a immobilized gel was prepared. 0.1 mL of the prepared FcR5a-immobilized gel was loaded on a spin column.
(2) The column packed with the FcR5a-immobilized gel prepared in (1) was washed three times with 0.5 mL of 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride.
(3) Human IgG1 (Sigma: I5154-1MG) or human as opposed to a medium in which 10% fetal bovine serum (FCS) is added to DMEM / F12 medium (manufactured by Life Technology), which is a medium for animal cells A simulated culture fluid was prepared by adding IgG3 (manufactured by Sigma: I 4639-1MG) to a concentration of 0.3 mg / mL.
(4) 0.4 mL of the mock culture solution prepared in (3) was added to the column washed with (2), and shaken at 25 ° C. for 2 hours.
(5) Remove the mock culture solution and wash four times with 0.5 mL of 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, and then 500 μL of 0.1 M glycine-hydrochloride buffer (pH 3.0) And eluted fractions every 100 μl.
(6) Mock culture broth, eluted fraction: 2 × sample buffer (2 (w / v)% sodium dodecyl sulfate, 6 (w / v)% β-mercaptoethanol, 10 (w / v)% glycerol and 0. Heat treatment was performed by adding 50 mM Tris-HCl buffer (pH 6.8) containing 005 (w / v)% bromophenol blue. Thereafter, the treated sample was separated by electrophoresis using a 5 to 20% gradient SDS-PAGE gel (manufactured by Atto Co., Ltd.). As a comparison, human IgG1 and human IgG3 (concentration: 0.2 mg / mL) added to the mock culture solution were similarly treated and analyzed by SDS-PAGE.

  The analysis result by SDS-PAGE of the eluted fraction containing human IgG1 or human IgG3 is shown in FIG. The eluted fraction containing human IgG1 obtained by purifying the mock culture solution to which human IgG1 has been added shows the same position as human IgG1 added to the mock culture solution, and the albumin which can be seen in the mock culture solution Since no band is observed, it can be confirmed that human IgG1 is purified with high purity (FIG. 6 (A)). In addition, the eluted fraction containing human IgG3 obtained by purifying the mock culture solution to which human IgG3 has been added shows the same position as human IgG3 added to the mock culture solution, and can be seen in the mock culture solution Since no albumin band is observed, it can be confirmed that human IgG3 is purified with high purity (FIG. 6 (B)). From these results, it was shown that FcR5a-immobilized gel, which is one aspect of the adsorbent of the present invention, can purify human IgG1 and human IgG3 from a culture solution for animal cells.

Claims (13)

In the amino acid sequence consisting of amino acid residues from 17 th to 192 th of the amino acid sequence shown in SEQ ID NO: 1, amino acid amino acid substitutions of at least (5) occurs among the following (1) (40) Human Fc binding protein , comprising a sequence .
(1) The 18th methionine of SEQ ID NO: 1 is substituted with arginine (2) The 27th valine of SEQ ID NO: 1 is substituted with glutamic acid (3) The 29th phenylalanine of SEQ ID NO: 1 is substituted with leucine or serine (4) 30 th leucine is replaced by the 35th tyrosine there asparagine or substituted proline (6) 46th lysine isoleucine or threonine of SEQ ID NO: 1 replaced with glutamine (5) SEQ ID NO: 1 SEQ ID NO: 1 (7 ) The 48th glutamine of SEQ ID NO: 1 is substituted with histidine or leucine (8) The 50th alanine of SEQ ID NO: 1 is substituted with histidine (9) The 51st tyrosine of SEQ ID NO: 1 is substituted with aspartic acid or histidine (10) ) The glutamic acid at position 54 of SEQ ID NO: 1 is substituted with aspartic acid or glycine (11) SEQ ID NO: The 56th asparagine of 1 is substituted with threonine (12) The 59th glutamine of SEQ ID NO: 1 is substituted with arginine (13) The 61st phenylalanine of SEQ ID NO: 1 is substituted with tyrosine (14) The 64th of SEQ ID NO: 1 Glutamate is substituted with aspartic acid (15) Serine at position 65 of SEQ ID NO: 1 is substituted with arginine (16) Alanine at position 71 of SEQ ID NO: 1 is substituted with aspartic acid (17) Phenylalanine at position 75 of SEQ ID NO: 1 is leucine (18) Aspartic acid of SEQ ID NO: 1 is substituted with asparagine (19) Alanine of 78th of SEQ ID NO: 1 is substituted with serine (20) 82th of SEQ ID NO: 1 Aspartate is replaced with glutamate or valine (21) 90th glutamine of SEQ ID NO: 1 Substitution of Arginine (22) Asparagine at 92nd in SEQ ID NO: 1 to Serine (23) Substitution of Leucine at 93rd of SEQ ID No. 1 to Arginine or Methionine (24) Substitution of 95th Threonine of SEQ ID NO: 1 to Alanine or Serine (25) leucine at position 110 of SEQ ID NO: 1 is substituted with glutamine (26) arginine at position 115 of SEQ ID NO: 1 is substituted for glutamine (27) tryptophan at position 116 of SEQ ID NO: 1 is substituted for leucine (28) Substitution of phenylalanine at position 118 of SEQ ID NO: 1 with tyrosine (29) Substitution of lysine at position 119 of SEQ ID NO: 1 with glutamic acid (30) Substitution of glutamic acid at position 120 of SEQ ID NO: 1 with valine (31) 121 of SEQ ID NO: 1 Glutamic acid is substituted with aspartic acid or glycine (32) Substitution of phenylalanine at position 151 in serine No. 1 to serine or tyrosine (33) substitution of serine at position 155 in SEQ ID NO: 1 to threonine (34) substitution of threonine at position 163 in SEQ ID NO: 1 to serine (35) in SEQ ID NO: 1 The 167th serine is substituted with glycine (36) The 169th serine of SEQ ID NO: 1 is substituted with a glycine (37) The 171st phenylalanine of SEQ ID NO: 1 is substituted with a tyrosine (38) The 180th asparagine of SEQ ID NO: 1 Substitution with any of lysine, serine or isoleucine (39) Substitution of threonine at position 185 of SEQ ID NO: 1 with serine (40) Substitution of glutamine at position 192 of SEQ ID NO: 1 with lysine SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 4
3, and 33 to 2 of the amino acid sequences described in any of SEQ ID NO: 47 and SEQ ID NO: 49
The human Fc binding protein according to claim 1 , comprising the amino acid residues up to the 08th position. The human Fc according to claim 2 , comprising the amino acid sequence set forth in any of SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 49. Binding protein. Furthermore, the human Fc binding protein according to any one of claims 1 to 3, wherein at least one of the following (41) to (44) amino acid substitution has occurred.
(41) leucine at position 66 of SEQ ID NO: 1 is substituted with histidine or arginine (42), glycine at position 147 of SEQ ID NO: 1 is substituted for aspartic acid (43), tyrosine at position 158 of SEQ ID NO: 1 is substituted for histidine (44) ) The valine at position 176 of SEQ ID NO: 1 is substituted with phenylalanine An adsorbent obtained by immobilizing the human Fc binding protein according to any one of claims 1 to 4 on an insoluble carrier. The purification method of the antibody using the adsorption agent of Claim 5 . A method for identifying differences in sugar chain structure possessed by antibodies by separating antibodies using the adsorbent according to claim 5 . The separation | isolation method of the sugar_chain | carbohydrate using the adsorption agent of Claim 5 . A polynucleotide encoding the human Fc binding protein according to any one of claims 1 to 4 . An expression vector comprising the polynucleotide according to claim 9 . A transformant obtained by transforming a host with the expression vector according to claim 10 . The transformant according to claim 11 , wherein the host is E. coli. To express the human Fc binding protein by culturing the transformant according to claim 11 or 12, to recover the human Fc binding protein expressed from the culture method for producing a human Fc binding proteins.

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